Pcv2 immunogenic compositions and methods of producing such compositions

ABSTRACT

An improved method for recovering the protein expressed by open reading frame 2 from porcine circovirus type 2 is provided. The method generally involves the steps of transfecting recombinant virus containing open reading frame 2 coding sequences into cells contained in growth media, causing the virus to express open reading frame 2, and recovering the expressed protein in the supernate. This recovery should take place beginning approximately 5 days after infection of the cells in order to permit sufficient quantities of recombinant protein to be expressed and secreted from the cell into the growth media. Such methods avoid costly and time consuming extraction procedures required to separate and recover the recombinant protein from within the cells.

SEQUENCE LISTING

This application contains a sequence listing in paper format and incomputer readable format, the teachings and content of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

One aspect of the present invention is concerned with the recovery of aprotein expressed by open reading frame 2 (ORF2) of porcine circovirustype 2 (PCV2). More particularly, the protein is a recombinant proteinexpressed by a transfected virus containing recombinant coding sequencesfor porcine circovirus type 2, open reading frame 2. Still moreparticularly, the transfected virus is permitted to infect cells ingrowth media and the protein expressed by open reading frame 2 isrecovered in the supernate, rather than from inside the cells. Even moreparticularly, the method involves the steps of amplifying the openreading frame 2 gene from porcine circovirus type 2, cloning thisamplified portion into a first vector, excising the open reading frame 2portion from this first vector and cloning it into a transfer vector,cotransfecting the transfer vector with a viral vector into cells ingrowth media, causing the cells to become infected by the viral vectorand thereby express open reading frame 2, and recovering the expressedrecombinant protein coded for by open reading frame 2 in the supernate.

In another aspect, the present invention is concerned with animmunogenic composition effective for inducing an immune responseagainst PCV2, and methods for producing those immunogenic compositions.More particularly, the present invention is concerned with animmunological composition effective for providing an immune responsethat protects an animal receiving the composition and reduces, orlessens the severity, of the clinical symptoms associated with PCV2infection. Still more particularly, the present invention is concernedwith a protein-based immunological composition that confers effectiveprotection against infection by PCV2. Even more particularly, thepresent invention is concerned with an immunological compositioncomprising ORF2 of PCV2, wherein administration of PCV2-ORF2 results inprotection against infection by PCV2. Most particularly, the presentinvention is concerned with an immunological composition effective forconferring effective immunity to a swine receiving the immunologicalcomposition, and wherein the composition comprises the protein expressedby ORF2 of PCV2.

2. Description of the Prior Art

Porcine circovirus type 2 (PCV2) is a small (17-22 nm in diameter),icosahedral, non-enveloped DNA virus, which contains a single-strandedcircular genome. PCV2 shares approximately 80% sequence identity withporcine circovirus type 1 (PCV1). However, in contrast with PCV1, whichis generally non-virulent, swine infected with PCV2 exhibit a syndromecommonly referred to as Post-weaning Multisystemic Wasting Syndrome(PMWS). PMWS is clinically characterized by wasting, paleness of theskin, unthriftiness, respiratory distress, diarrhea, icterus, andjaundice. In some affected swine, a combination of all symptoms will beapparent while other swine will only have one or two of these symptoms.During necropsy, microscopic and macroscopic lesions also appear onmultiple tissues and organs, with lymphoid organs being the most commonsite for lesions. A strong correlation has been observed between theamount of PCV2 nucleic acid or antigen and the severity of microscopiclymphoid lesions. Mortality rates for swine infected with PCV2 canapproach 80%. In addition to PMWS, PCV2 has been associated with severalother infections including pseudorabies, porcine reproductive andrespiratory syndrome (PRRS), Glasser's disease, streptococcalmeningitis, salmonellosis, postweaning colibacillosis, dietetichepatosis, and suppurative bronchopneumonia.

Open reading frame 2 (ORF2) protein of PCV2, having an approximatemolecular weight of 30 kDa when run on SDS-PAGE gel, has been utilizedin the past as an antigenic component in vaccines for PCV2. Typicalmethods of obtaining ORF2 for use in such vaccines generally consist ofamplifying the PCV2 DNA coding for ORF2, transfecting a viral vectorwith the ORF2 DNA, infecting cells with the viral vector containing theORF2 DNA, permitting the virus to express ORF2 protein within the cell,and extracting the ORF2 protein from the cell via cell lysis. Theseprocedures generally take up to about four days after infection of thecells by the viral vector. However, these procedures have a disadvantagein that the extraction procedures are both costly and time-consuming.Additionally, the amount of ORF2 recovered from the cells is not veryhigh; consequently, a large number of cells need to be infected by alarge number of viral vectors in order to obtain sufficient quantitiesof the recombinant expressed protein for use in vaccines and the like.

Current approaches to PCV2 immunization include DNA-based vaccines, suchas those described in U.S. Pat. No. 6,703,023. However, such vaccineshave been ineffective at conferring protective immunity against PCV2infection and the clinical signs associated therewith.

Accordingly, what is needed in the art is a method of obtaining ORF2protein, which does not require extraction of the ORF2 protein fromwithin infected cells. What is further needed are methods of obtainingrecombinant ORF2 protein in quantities sufficient for efficientlypreparing vaccine compositions. What is still further needed are methodsfor obtaining ORF2 protein which do not require the complicated andlabor-intensive methods required by the current ORF2 protein extractionprotocols. Finally, with respect to compositions, what is needed in theart is an immunogenic composition which does confer protective immunityagainst PCV2 infection and lessens the severity of or prevents theclinical signs associated therewith.

SUMMARY OF THE INVENTION

The present invention overcomes the problems inherent in the prior artand provides a distinct advance in the state of the art. Specifically,one aspect of the present invention provides improved methods ofproducing and/or recovering recombinant PCV2 ORF2 protein, i) bypermitting infection of susceptible cells in culture with a recombinantviral vector containing PCV2 ORF2 DNA coding sequences, wherein ORF2protein is expressed by the recombinant viral vector, and ii) thereafterrecovering the ORF2 in the supernate. It has been unexpectedlydiscovered that ORF2 is released into the supernate in large quantitiesif the infection and subsequent incubation of the infected cells isallowed to progress past the typical prior PCV 2 ORF2 recovery process,which extracts the PCV2 ORF2 from within cells. It furthermore has beensurprisingly found, that PCV ORF2 protein is robust against prototypicaldegradation outside of the production cells. Both findings togetherallow a recovery of high amounts of PCV2 ORF2 protein from the supernateof cell cultures infected with recombinant viral vectors containing aPCV2 ORF2 DNA and expressing the PCV2 ORF2 protein. High amounts of PCV2ORF2 protein means more than about 20 μg/mL supernate, preferably morethan about 25 μg/mL, even more preferred more than about 30 μg/mL, evenmore preferred more than about 40 μg/mL, even more preferred more thanabout 50 μg/mL, even more preferred more than about 60 μg/mL, even morepreferred more than about 80 μg/mL, even more preferred more than about100 μg/mL, even more preferred than about 150 μg/mL, most preferred thanabout 190 μg/mL. Those expression rates can also be achieved for exampleby the methods as described in Examples 1 to 3.

Preferred cell cultures have a cell count between about 0.3-2.0×10⁶cells/mL, more preferably from about 0.35-1.9×10⁶ cells/mL, still morepreferably from about 0.4-1.8×10⁶ cells/mL, even more preferably fromabout 0.45-1.7×10⁶ cells/mL, and most preferably from about 0.5-1.5×10⁶cells/mL. Preferred cells are determinable by those of skill in the art.Preferred cells are those susceptible for infection with an appropriaterecombinant viral vector, containing a PCV2 ORF2 DNA and expressing thePCV2 ORF2 protein. Preferably the cells are insect cells, and morepreferably, they include the insect cells sold under the trademark Sf+insect cells (Protein Sciences Corporation, Meriden, Conn.).

Appropriate growth media will also be determinable by those of skill inthe art with a preferred growth media being serum-free insect cell mediasuch as Excell 420 (JRH Biosciences, Inc., Lenexa, Kans.) and the like.Preferred viral vectors include baculovirus such as BaculoGold (BDBiosciences Pharmingen, San Diego, Calif.), in particular if theproduction cells are insect cells. Although the baculovirus expressionsystem is preferred, it is understood by those of skill in the art thatother expression systems will work for purposes of the presentinvention, namely the expression of PCV2 ORF2 into the supernatant of acell culture. Such other expression systems may require the use of asignal sequence in order to cause ORF2 expression into the media. It hasbeen surprisingly discovered that when ORF2 is produced by a baculovirusexpression system, it does not require any signal sequence or furthermodification to cause expression of ORF2 into the media. It is believedthat this protein can independently form virus-like particles (Journalof General Virology Vol. 81, pp. 2281-2287 (2000) and be secreted intothe culture supernate. The recombinant viral vector containing the PCV2ORF2 DNA sequences has a preferred multiplicity of infection (MOI) ofbetween about 0.03-1.5, more preferably from about 0.05-1.3, still morepreferably from about 0.09-1.1, and most preferably from about 0.1-1.0,when used for the infection of the susceptible cells. Preferably theMOIs mentioned above relates to one mL of cell culture fluid.Preferably, the method described herein comprises the infection of0.35-1.9×10⁶ cells/mL, still more preferably of about 0.4-1.8×10⁶cells/mL, even more preferably of about 0.45-1.7×10⁶ cells/mL, and mostpreferably of about 0.5-1.5×10⁶ cells/mL with a recombinant viral vectorcontaining a PCV2 ORF2 DNA and expressing the PCV2 ORF protein having aMOI (multiplicity of infection) of between about 0.03-1.5, morepreferably from about 0.05-1.3, still more preferably from about0.09-1.1, and most preferably from about 0.1-1.0.

The infected cells are then incubated over a period of up to ten days,more preferably from about two days to about ten days, still morepreferably from about four days to about nine days, and most preferablyfrom about five days to about eight days. Preferred incubationconditions include a temperature between about 22-32° C., morepreferably from about 24-30° C., still more preferably from about 25-29°C., even more preferably from about 26-28° C., and most preferably about27° C. Preferably, the Sf+ cells are observed following inoculation forcharacteristic baculovirus-induced changes. Such observation may includemonitoring cell density trends and the decrease in viability during thepost-infection period. It was found that peak viral titer is observed3-5 days after infection and peak ORF2 release from the cells into thesupernate is obtained between days 5 and 8, and/or when cell viabilitydecreases to less than 10%.

Thus, one aspect of the present invention provides an improved method ofproducing and/or recovering recombinant PCV2 ORF2 protein, preferably inamounts described above, by i) permitting infection of a number ofsusceptible cells (see above) in culture with a recombinant viral vectorwith a MOI as defined above, ii) expressing PCV2 ORF2 protein by therecombinant viral vector, and iii) thereafter recovering the PCV2 ORF2in the supernate of cells obtained between days 5 and 8 after infectionand/or cell viability decreases to less then 10%. Preferably, therecombinant viral vector is a recombinant baculovirus containing PCV2ORF2 DNA coding sequences and the cells are Sf+ cells. Additionally, itis preferred that the culture be periodically examined for macroscopicand microscopic evidence of contamination or for atypical changes incell morphology during the post-infection period. Any culture exhibitingany contamination should be discarded. Preferably, the expressed ORF2recombinant protein is secreted by the cells into the surrounding growthmedia that maintains cell viability. The ORF2 is then recovered in thesupernate surrounding the cells rather than from the cells themselves.

The recovery process preferably begins with the separation of celldebris from the expressed ORF2 in media via a separation step. Preferredseparation steps include filtration, centrifugation at speeds up toabout 20,000×g, continuous flow centrifugation, chromatographicseparation using ion exchange or gel filtration, and conventionalimmunoaffinity methods. Those methods are known to persons skilled inthe art for example by (Harris and Angel (eds.), Protein purificationmethods—a practical approach, IRL press Oxford 1995). The most preferredseparation methods include centrifugation at speeds up to about 20,000×gand filtration. Preferred filtration methods include dead-endmicrofiltration and tangential flow (or cross flow) filtration includinghollow fiber filtration dead-end micro filtration. Of these, dead-endmicrofiltration is preferred. Preferred pore sizes for dead-endmicrofiltration are between about 0.30-1.35 μm, more preferably betweenabout 0.35-1.25 μm, still more preferably between about 0.40-1.10 μm,and most preferably between about 0.45-1.0 μm. It is believed that anyconventional filtration membrane will work for purposes of the presentinvention and polyethersulfone membranes are preferred. Any low weightnucleic acid species are removed during the filtration step.

Thus, one further aspect of the present invention provides an improvedmethod of producing and/or recovering recombinant PCV2 ORF2 protein,preferably in amounts described above, by i) permitting infection of anumber of susceptible cells (see above) in culture with a recombinantviral vector with a MOI as defined above, ii) expressing PCV ORF2protein by the recombinant viral vector, iii) recovering the PCV2 ORF2in the supernate of cells obtained between days 5 and 8 after infectionand/or cell viability decreases to less then 10%, and, iv) separatingcell debris from the expressed PCV2 ORF2 via a separation step.Preferably, the recombinant viral vector is a baculovirus containingORF2 DNA coding sequences and the cells are SF+ cells. Preferredseparation steps are those described above. Most preferred is a dead-endmicrofiltration using a membrane having a pore size between about0.30-1.35 μm, more preferably between about 0.35-1.25 μm, still morepreferably between about 0.40-1.10 μm, and most preferably between about0.45-1.0 μm.

For recovery of PCV2 ORF2 that will be used in an immunogenic orimmunological composition such as a vaccine, the inclusion of aninactivation step is preferred in order to inactivate the viral vector.An “immunogenic or immunological composition” refers to a composition ofmatter that comprises at least one antigen which elicits animmunological response in the host of a cellular and/orantibody-mediated immune response to the composition or vaccine ofinterest. Usually, an “immunological response” includes but is notlimited to one or more of the following effects: the production oractivation of antibodies, B cells, helper T cells, suppressor T cells,and/or cytotoxic T cells and/or yd T cells, directed specifically to anantigen or antigens included in the composition or vaccine of interest.Preferably, the host will display either a therapeutic or protectiveimmunological response such that resistance to new infection will beenhanced and/or the clinical severity of the disease reduced. Suchprotection will be demonstrated by either a reduction or lack ofsymptoms normally displayed by an infected host, a quicker recovery timeand/or a lowered viral titer in the infected host. Thus, the presentinvention also relates to method of producing and/or recoveringrecombinant PCV2 ORF2 protein, preferably in amounts described above, byi) permitting infection of a number of susceptible cells (see above) inculture with a recombinant viral vector with a MOI as defined above, ii)expressing PCV ORF2 protein by the recombinant viral vector, iii)recovering the PCV2 ORF2 in the supernate of cells obtained between days5 and 8 after infection and/or cell viability decreases to less then10%, iv) separating cell debris from the expressed PCV2 ORF2 via aseparation step, and v) inactivating the recombinant viral vector.

Preferably, this inactivation is done either just before or just afterthe filtration step, with after the filtration step being the preferredtime for inactivation. Any conventional inactivation method can be usedfor purposes of the present invention. Thus, inactivation can beperformed by chemical and/or physical treatments. In preferred forms,the volume of harvest fluids is determined and the temperature isbrought to between about 32-42° C., more preferably between about 34-40°C., and most preferably between about 35-39° C. Preferred inactivationmethods include the addition cyclized binary ethylenimine (BEI),preferably in a concentration of about 1 to about 20 mM, preferably ofabout 2 to about 10 mM, still more preferably of about 2 to about 8 mM,still more preferably of about 3 to about 7 mM, most preferably of about5 mM. For example the inactivation includes the addition of a solutionof 2-bromoethyleneamine hydrobromide, preferably of about 0.4M, whichhas been cyclized to 0.2M binary ethylenimine (BEI) in 0.3N NaOH, to thefluids to give a final concentration of about 5 mM BEI. Preferably, thefluids are then stirred continuously for 72-96 hours and the inactivatedharvest fluids can be stored frozen at −40° C. or below or between about1-7° C. After inactivation is completed a sodium thiosulfate solution,preferably at 1.0M is added to neutralize any residual BEI. Preferably,the sodium thiosulfate is added in equivalent amount as compared to theBEI added prior to for inactivation. For example, in the event BEI isadded to a final concentration of 5 mM, a 1.0M sodium thiosulfatesolution is added to give a final minimum concentration of 5 mM toneutralize any residual BEI.

Thus, one further aspect of the present invention relates to a method ofproducing recombinant PCV2 ORF2 protein, preferably in amounts describedabove, by i) permitting infection of a number of susceptible cells (seeabove) in culture with a recombinant viral vector with a MOI as definedabove, ii) expressing PCV ORF2 protein by the recombinant viral vector,iii) recovering the PCV2 ORF2 in the supernate of cells obtained betweendays 5 and 8 after infection and/or cell viability decreases to lessthen 10%, iv) separating cell debris from the expressed PCV2 ORF2 via aseparation step, and v) inactivating the recombinant viral vector.Preferably, the recombinant viral vector is a baculovirus containingORF2 DNA coding sequences and the cells are SF+ cells. Preferredseparation steps are those described above, most preferred is thefiltration step. Preferred inactivation steps are those described above.Preferably, inactivation is performed between about 35-39° C. and in thepresence of 2 to 8 mM BEI, still more preferred in the presence of about5 mM BEI. It has been surprisingly found, that higher concentrations ofBEI negatively affect the PCV2 ORF2 protein.

According to one further aspect of the present invention, the methoddescribed above also includes an neutralization step after step v). Thisstep vi) comprises adding of an equivalent amount of an agent thatneutralizes the inactivation agent within the solution. Preferably, ifthe inactivation agent is BEI, addition of sodium thiosulfate to anequivalent amount is preferred. Thus, according to a further aspect,step vi) comprises adding of a sodium thiosulfate solution to a finalconcentration of about 1 to about 20 mM, preferably of about 2 to about10 mM, still more preferably of about 2 to about 8 mM, still morepreferably of about 3 to about 7 mM most preferably of about 5 mM, whenthe inactivation agent is BEI.

In preferred forms and especially in forms that will use the recombinantPCV2 ORF2 protein in an immunogenic composition such as a vaccine, eachlot of harvested ORF2 will be tested for inactivation by passage in theanchorage dependent, baculovirus susceptible Sf+ cells. In a preferredform of this testing, 150 cm² of appropriate cell culture monolayer isinoculated with 1.0 mL of inactivated PCV2 fluids and maintained at25-29° C. for 14 days with at least two passages. At the end of themaintenance period, the cell monolayers are examined for cytopathogeniceffect (CPE) typical of PCV2 ORF2 baculovirus. Preferably, positivevirus controls are also used. Such controls can consist of one cultureof Sf+ cells inoculated with a non-inactivated reference PCV2 ORF2baculovirus and one flask of Sf+ cells that remain uninoculated. Afterincubation and passage, the absence of virus-infected cells in the BEItreated viral fluids would constitute a satisfactory inactivation test.The control cells inoculated with the reference virus should exhibit CPEtypical of PCV2 ORF2 baculovirus and the uninoculated flask should notexhibit any evidence of PCV2 ORF2 baculovirus CPE. Alternatively, at theend of the maintenance period, the supernatant samples could becollected and inoculated onto a Sf+ 96 well plate, which has been loadedwith Sf+ cells, and then maintained at 25-29° C. for 5-6 days. The plateis then fixed and stained with anti-PCV2 ORF2 antibody conjugated toFITC. The absence of CPE and ORF2 expression, as detected by IFAmicoscopy, in the BEI treated viral fluids constitutes a satisfactoryinactivation test. The control cells inoculated with the reference virusshould exhibit CPE and IFA activity and the uninoculated flask shouldnot exhibit any evidence of PCV2 ORF2 baculovirus CPE and contain no IFAactivity.

Thus a further aspect of the present invention relates to aninactivation test for determining the effectiveness of the inactivationof the recombination viral vector, comprises the steps: i) contacting atleast a portion of the culture fluid containing the recombinant viralvector with an inactivating agent, preferably as described above, ii)adding a neutralization agent to neutralize the inactivation agent,preferably as described above, and iii) determining the residualinfectivity by the assays as described above.

After inactivation, the relative amount of recombinant PCV2 ORF2 proteinin a sample can be determined in a number of ways. Preferred methods ofquantitation include SDS-PAGE densitometry, ELISA, and animalvaccination studies that correlate known quantities of vaccine withclinical outcomes (serology, etc.). When SDS-PAGE is utilized forquantitation, the sample material containing an unknown amount ofrecombinant PCV2 ORF2 protein is run on a gel, together with samplesthat contain different known amounts of recombinant PCV2 ORF2 protein. Astandard curve can then be produced based on the known samples and theamount of recombinant PCV2 ORF2 in the unknown sample can be determinedby comparison with this standard curve. Because ELISAs are generallyrecognized as the industry standard for antigen quantitation, they arepreferred for quantitation.

Thus, according to a further aspect, the present invention also relatesto an ELISA for the quantification of recombinant PCV2 ORF2 protein. Apreferred ELISA as provided herewith will generally begin with dilutingthe capture antibody 1:6000 or an appropriate working dilution incoating buffer. A preferred capture antibody is Swine anti-PCV2 PAbProt. G purified, and a preferred coating buffer is 0.05M Carbonatebuffer, which can be made by combining 2.93 g NaHCO₃ (Sigma Cat. No.S-6014, or equivalent) and 1.59 g NaCO₃ (Sigma Cat. No. S-6139, orequivalent). The mixture is combined with distilled water, orequivalent, to make one liter at a pH of 9.6±0.1. Next, the captureantibody is diluted 1:6000, or any other appropriate working dilution,in coating buffer. For example, for four plates, one would need 42 mLsof coating buffer and seven μL of capture antibody. Using a reversepipetting method, 100 μL of diluted capture antibody is added to all ofthe wells. In order to obtain an even coating, the sides of each plateshould be gently tapped. The plates are then sealed with plate sealers,prior to stacking the plates and capping the stack with an empty 96-wellplate. The plates are incubated overnight (14-24 hours) at 35-39° C.Each plate is then washed three times with wash buffer using the ultrawash plus micro titer plate washer set at 250 μL/wash with three washesand no soak time. After the last wash, the plates should be tapped ontoa paper towel. Again, using the reverse pipetting technique, 250 μL ofblocking solution should be added to all of the wells. The test platesshould be sealed and incubated for approximately one hour (±fiveminutes) at 35-37° C. Preferably, the plates will not be stacked afterthis step. During the blocking step, all test samples should be pulledout and thawed at room temperature. Next, four separate dilution platesshould be prepared by adding 200 μL of diluent solution to all of theremaining wells except for row A and row H, columns 1-3. Next, six testtubes should be labeled as follows, low titer, medium titer, high titer,inactivated/filtered (1:240), inactivated/filtered (1:480), and internalcontrol. In the designated tubes, an appropriate dilution should beprepared for the following test samples. The thawed test samples shouldbe vortexed prior to use. For four plates, the following dilutions willbe made: A) the low titer will not be pre-diluted: 3.0 mLs of low titer;B) negative control at a 1:30 dilution (SF+ cells): 3.77 mLs ofdiluent+130 μL of the negative control; C) medium titer at a 1:30dilution (8 μg/mL): 3.77 mLs of diluent+130 μL of the medium titer; D)high titer at a 1:90 dilution (16 μg/mL): 2.967 mLs of diluent+33 μL ofhigh titer; E) inactivated/filtered at a 1:240 dilution: 2.39 mLs ofdiluent+10 μL of inactivated/filtered sample; F) inactivated/filtered ata 1:480 dilution: 1.0 mL of diluent+1.0 mL of inact/filtered (1:240)prepared sample from E above; G) internal control at 1:30 dilution: 3.77mLs of diluent+130 μL of the internal control. Next, add 300 μL of theprepared samples to corresponding empty wells in the dilution plates forplates 1 through 4. The multichannel pipettor is then set to 100 μL andthe contents in Row A are mixed by pipetting up and down for at least 5times and then 100 μL is transferred to Row B using the reversepipetting technique. The tips should be changed and this same procedureis followed down the plate to Row G. Samples in these dilution platesare now ready for transfer to the test plates once the test plates havebeen washed 3 times with wash buffer using the ultrawash plus microtiterplate washer (settings at 250 μL/wash, 3 washes, no soak time). Afterthe last wash, the plates should be tapped onto a paper towel. Next, thecontents of the dilution plate are transferred to the test plate using asimple transfer procedure. More specifically, starting at row H, 100μL/well is transferred from the dilution plate(s) to corresponding wellsof the test plate(s) using reverse pipetting technique. After eachtransfer, the pipette tips should be changed. From Row G, 100 μL/well inthe dilution plate(s) is transferred to corresponding wells of the testplate(s) using reverse pipetting technique. The same set of pipette tipscan be used for the remaining transfer. To ensure a homogenous solutionfor the transfer, the solution should be pipetted up and down at least 3times prior to transfer. Next, the test plate(s) are sealed andincubated for 1.0 hour±5 minutes at 37° C.±2.0° C. Again, it ispreferable not to stack the plates. The plates are then washed 3 timeswith wash buffer using the ultrawash plus microtiter plate washer(settings at 250 μL/wash, 3 washes, and no soak time). After the lastwash, the plates are tapped onto a paper towel. Using reverse pipettingtechnique, 100 μL of detection antibody diluted 1:300, or appropriateworking dilution, in diluent solution is added to all of the wells ofthe test plate(s). For example, for four plates, one will need 42 mLs ofdiluent solution with 140 μL of capture antibody. The test plate(s) arethen sealed and incubated for 1.0 hour±5 minutes at 37° C.±2.0° C.Again, the plates are washed 3 times with wash buffer using theultrawash plus microtiter plate washer (settings at 250 μL/wash, 3washes, and no soak time). After the last wash, the plates are tappedonto a paper towel. Next, the conjugate diluent is prepared by adding 1%normal rabbit serum to the diluent. For example, for four plates, 420 μLof normal rabbit serum is added to 42 mL of diluent. The conjugateantibody is diluted to 1:10,000, or any other appropriate workingdilution, in a freshly prepared conjugate diluent solution to all wellsof the test plate(s). Using a reverse pipetting technique, 100 μL ofthis diluted conjugate antibody is added to all the wells. The testplate(s) are then sealed and incubated for 45±5 minutes at 37° C.±2.0°C. Preferably, the plates are not stacked. The plates are then washed 3times with wash buffer using the ultrawash plus microtiter plate washer(settings at 250 μL/wash, 3 washes, and no soak time). After the lastwash, the plates are tapped onto a paper towel. Next, equal volumes ofTMB Peroxidase Substrate (Reagent A) with Peroxidase Solution B (ReagentB) are mixed immediately prior to use. The amount mixed will varydepending upon the quantity of plates but each plate will require 10mL/plate+2 mLs. Therefore, for 4 plates, it will be 21 mL of ReagentA+21 mL of Reagent B. Using a reverse pipetting technique, 100 μL ofsubstrate is added to all wells of the test plate(s). The plates arethen incubated at room temperature for 15 minutes±15 seconds. Thereaction is stopped by the addition of 100 μL of 1N HCl solution to allwells using a reverse pipetting technique. The ELISA plate reader isthen turned on and allowed to proceed through its diagnostics andtesting phases in a conventional manner.

A further aspect of the invention relates to a method for constructing arecombinant viral vector containing PCV2 ORF2 DNA and expressing PCV2ORF2 protein in high amounts, when infected into susceptible cells. Ithas been surprisingly found that the recombinant viral vector asprovided herewith expresses high amounts, as defined above, of PCV2 ORF2after infecting susceptible cells. Therefore, the present invention alsorelates to an improved method for producing and/or recovering of PCV2ORF2 protein, preferably comprises the step: constructing a recombinantviral vector containing PCV2 ORF2 DNA and expressing PCV2 ORF2 protein.Preferably, the viral vector is a recombinant baculovirus. Details ofthe method for constructing recombinant viral vectors containing PCV2ORF2 DNA and expressing PCV2 ORF2 protein, as provided herewith, aredescribed to the following: In preferred forms the recombinant viralvector containing PCV2 ORF 2 DNA and expressing PCV2 ORF2 protein usedto infect the cells is generated by transfecting a transfer vector thathas had an ORF2 gene cloned therein into a viral vector. Preferably,only the portion of the transfer vector is transfected into the viralvector, that contains the ORF2 DNA. The term “transfected into a viralvector” means, and is used as a synonym for “introducing” or “cloning” aheterologous DNA into a viral vector, such as for example into abaculovirus vector. The viral vector is preferably but not necessarily abaculovirus.

Thus, according to a further aspect of the present invention, therecombinant viral vector is generated by recombination between atransfer vector containing the heterologous PCV2 ORF2 DNA and a viralvector, preferably a baculovirus, even more preferably a linearizedreplication-deficient baculovirus (such as Baculo Gold DNA). A “transfervector” means a DNA molecule, that includes at least one origin ofreplication, the heterologous gene, in the present case PCV2 ORF2, andDNA sequences which allows the cloning of said heterologous gene intothe viral vector. Preferably the sequences which allow cloning of theheterologous gene into the viral vector are flanking the heterologousgene. Even more preferably those flanking sequences are at leasthomologous in parts with sequences of the viral vector. The sequencehomology then allows recombination of both molecules, the viral vectorand the transfer vector to generate a recombinant viral vectorcontaining the heterologous gene. One preferred transfer vector is thepVL1392 vector (BD Biosciences Pharmingen), which is designed forco-transfection with the BaculoGold DNA into the preferred Sf+ cellline. Preferably, said transfer vector comprises a PCV2 ORF2 DNA. Theconstruct co-transfected is approximately 10,387 base pairs in length.

In more preferred forms, the methods of the present invention will beginwith the isolation of PCV2 ORF2 DNA. Generally, this can be from a knownor unknown strain as the ORF2 DNA appears to be highly conserved with atleast about 95% sequence identity between different isolates. Any PCV2ORF2 gene known in the art can be used for purposes of the presentinvention as each would be expressed into the supernate. The PCV ORF2DNA is preferably amplified using PCR methods, even more preferredtogether with the introduction of a 5′ flanking Kozak's consensussequence (CCGCCAUG) (SEQ ID NO 1) and/or a 3′ flanking EcoR1 site(GAATTC) (SEQ ID NO 2). Such introduction of a 5′ Kozak's consensuspreferably removes the naturally occurring start codon AUG of PCV2 ORF2.The 3′ EcoR1 site is preferably introduced downstream of the stop codonof the PCV2 ORF2. More preferably it is introduced downstream of a polyA transcription termination sequence, that itself is located downstreamof the PCV2 ORF2 stop codon. It has been found, that the use of a Kozakconsensus sequence, in particular as described above, increases theexpression level of the subsequent PCV2 ORF2 protein. The amplified PCV2ORF2 DNA, with these additional sequences, is cloned into a vector. Apreferred vector for this initial cloning step is the pGEM-T-Easy Vector(Promega, Madison, Wis.). The PCV2 ORF2 DNA including some pGEM vectorsequences (SEQ ID NO: 7) is preferably excised from the vector at theNot1 restriction site. The resulting DNA is then cloned into thetransfer vector.

Thus, in one aspect of the present invention, a method for constructinga recombinant viral vector containing PCV2 ORF2 DNA is provided. Thismethod comprises the steps: i) cloning a recombinant PCV2 ORF2 into atransfer vector; and ii) transfecting the portion of the transfer vectorcontaining the recombinant PCV2 ORF2 into a viral vector, to generatethe recombinant viral vector. Preferably, the transfer vector is thatdescribed above or is constructed as described above or as exemplarilyshown in FIG. 1. Thus according to a further aspect, the transfervector, used for the construction of the recombinant viral vector asdescribed herein, contains the sequence of SEQ ID NO: 7.

According to a further aspect, this method further comprises prior tostep i) the following step: amplifying the PCV2 ORF2 DNA in vitro,wherein the flanking sequences of the PCV2 ORF2 DNA are modified asdescribed above. In vitro methods for amplifying the PCV2 ORF2 DNA andmodifying the flanking sequences, cloning in vitro amplified PCV2 ORF2DNA into a transfer vector and suitable transfer vectors are describedabove, exemplarily shown in FIG. 1, or known to a person skilled in theart. Thus according to a further aspect, the present invention relatesto a method for constructing a recombinant viral vector containing PCV2ORF2 DNA and expressing PCV2 ORF2 protein comprises the steps of: i)amplifying PCV2 ORF2 DNA in vitro, wherein the flanking sequences ofsaid PCV2 ORF2 DNA are modified, ii) cloning the amplified PCV2 ORF2 DNAinto a transfer vector; and iii) transfecting the transfer vector or aportion thereof containing the recombinant PCV2 ORF2 DNA into a viralvector to generate the recombinant viral vector. Preferably, themodification of the flanking sequences of the PCV2 ORF2 DNA is performedas described above, e.g. by introducing a 5′ Kozak sequence and/or aEcoR 1 site, preferably as described above.

According to a further aspect, a method of producing and/or recoveringrecombinant protein expressed by open reading frame 2 of PCV2 isprovided. The method generally comprises the steps of: i) cloning arecombinant PCV2 ORF2 into a transfer vector; ii) transfecting theportion of the transfer vector containing the recombinant PCV2 ORF2 intoa virus; iii) infecting cells in media with the transfected virus; iv)causing the transfected virus to express the recombinant protein fromPCV2 ORF2; v) separating cells from the supernate; and vi) recoveringthe expressed PCV2 ORF2protein from the supernate.

Methods of how to clone a recombinant PCV2 ORF2 DNA into a transfervector are described above. Preferably, the transfer vector contains thesequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7. However, the transfervector can contain any PCV2 ORF2 DNA, unmodified or modified, as long asthe PCV2 ORF2 DNA, when transfected into a recombinant viral vector, isexpressed in cell culture. Preferably, the recombinant viral vectorcomprises the sequence of SEQ ID NO:8. Moreover, methods of how toinfect cells, preferably how to infect insect cells with a definednumber of recombinant baculovirus containing PCV2 ORF2 DNA andexpressing PCV2 ORF2 protein are described above in detail. Moreover,steps of separating cells from the supernate as well as steps forrecovering the expressed PCV2 ORF2 protein are also described above indetail. Any of these specific process steps, as described herein, arepart of the method of producing and/or recovering recombinant proteinexpressed by open reading frame 2 of PCV2 as described above.Preferably, the cells are SF+ cells. Still more preferably, cellcultures have a cell count between about 0.3-2.0×10⁶ cells/mL, morepreferably from about 0.35-1.9×10⁶ cells/mL, still more preferably fromabout 0.4-1.8×10⁶ cells/mL, even more preferably from about 0.45-1.7×10⁶cells/mL, and most preferably from about 0.5-1.5×10⁶ cells/mL.Preferably, the recombinant viral vector containing the PCV2 ORF2 DNAhas a preferred multiplicity of infection (MOI) of between about0.03-1.5, more preferably from about 0.05-1.3, still more preferablyfrom about 0.09-1.1, still more preferably from about 0.1-1.0, and mostpreferably to about 0.5, when used for the infection of the susceptiblecells. Preferably, recovering of the PCV2 ORF2 protein in the supernateof cells obtained between days 5 and 8 after infection and/or cellviability decreases to less then 10%. Preferably, for producing PCV2ORF2 protein, cells are cultivated at 25 to 29° C. Preferably, theseparation step is a centrifugation or a filtration step.

Optionally, this method can include the step of amplifying the PCV2 ORF2DNA from a strain of PCV2 prior to cloning the PCV2 ORF2 DNA into thetransfer vector. In preferred forms, a 5′ Kozak's sequence, a 3′ EcoR1site, and combinations thereof can also be added to the amplifiedsequence, preferably prior to or during amplification. A preferred 5′Kozak's sequence comprises SEQ ID NO: 1. A preferred 3′ EcoR1 sitecomprises SEQ ID NO: 2. Preferred PCV2 ORF2 DNA comprises the nucleotidesequence Genbank Accession No. AF086834 (SEQ ID NO: 3) and SEQ ID NO: 4.Preferred recombinant PCV2 ORF2 protein comprises the amino acidsequence of SEQ ID NO: 5, which is the protein encoded by SEQ ID NO: 3(Genbank Accession No. AF086834) and SEQ ID No: 6, which is the proteinencoded by SEQ ID NO: 4. A preferred media comprises serum-free insectcell media, still more preferably Excel 420 media. When the optionalamplification step is performed, it is preferable to first clone theamplified open reading frame 2 into a first vector, excise the openreading frame 2 from the first vector, and use the excised open readingframe for cloning into the transfer vector. A preferred cell line forcotransfection is the SF+ cell line. A preferred virus forcotransfection is baculovirus. In preferred forms of this method, thetransfected portion of the transfer vector comprises SEQ ID NO: 8.Finally, for this method, it is preferred to recover the PCV2 openreading frame 2 (ORF2) protein in the cell culture supernate at least 5days after infecting the cells with the virus.

Thus, a further aspect of the invention relates to a method forproducing and/or recovering the PCV2 open reading frame 2, comprises thesteps: i) amplifying the PCV2 ORF2 DNA in vitro, preferably by adding a5′ Kozak sequence and/or by adding a 3′ EcoR1 restriction site, ii)cloning the amplified PCV2 ORF2 into a transfer vector; iii)transfecting the portion of the transfer vector containing therecombinant PCV2 ORF2 into a virus; iv) infecting cells in media withthe transfected virus; v) causing the transfected virus to express therecombinant protein from PCV2 ORF2; vi) separating cells from thesupernate; and vii) recovering the expressed PCV2 ORF2 protein from thesupernate.

A further aspect of the present invention relates to a method forpreparing a composition comprising PCV2 ORF2 protein, and inactivatedviral vector. This method comprises the steps: i) cloning the amplifiedPCV2 ORF2 into a transfer vector; ii) transfecting the portion of thetransfer vector containing the recombinant PCV2 ORF2 into a virus; iii)infecting cells in media with the transfected viral vector; iv) causingthe transfected viral vector to express the recombinant protein fromPCV2 ORF2; v) separating cells from the supernate; vi) recovering theexpressed PCV2 ORF2 protein from the supernate; and vii) inactivatingthe recombinant viral vector. Preferably, the recombinant viral vectoris a baculovirus containing ORF2 DNA coding sequences and the cells areSF+ cells. Preferred separation steps are those described above, mostpreferred is the filtration step. Preferred inactivation steps are thosedescribed above. Preferably, inactivation is performed between about35-39° C. and in the presence of 2 to 8 mM BEI, still more preferred inthe presence of about 5 mM BEI. It has been surprisingly found, thathigher concentrations of BEI negatively affect the PCV2 ORF2 protein,and lower concentrations are not effective to inactivate the viralvector within 24 to 72 hours of inactivation. Preferably, inactivationis performed for at least 24 hours, even more preferred for 24 to 72hours.

According to a further aspect, the method for preparing a compositioncomprising PCV2 ORF2 protein, and inactivated viral vector, as describedabove, also includes an neutralization step after step vii). This stepviii) comprises adding of an equivalent amount of an agent thatneutralizes the inactivation agent within the solution. Preferably, ifthe inactivation agent is BEI, addition of sodium thiosulfate to anequivalent amount is preferred. Thus, according to a further aspect,step viii) comprises adding of a sodium thiosulfate solution to a finalconcentration of about 1 to about 20 mM, preferably of about 2 to about10 mM, still more preferably of about 2 to about 8 mM, still morepreferably of about 3 to about 7 mM, most preferably of about 5 mM, whenthe inactivation agent is BEI.

According to a further aspect, the method for preparing a compositioncomprising PCV2 ORF2 protein, and inactivated viral vector, as describedabove, comprises prior to step i) the following step: amplifying thePCV2 ORF2 DNA in vitro, wherein the flanking sequences of the PCV2 ORF2DNA are modified as described above. In vitro methods for amplifying thePCV2 ORF2 DNA and modifying the flanking sequences, cloning in vitroamplified PCV2 ORF2 DNA into a transfer vector and suitable transfervectors are described above, exemplarily shown in FIG. 1, or known to aperson skilled in the art. Thus according to a further aspect, thismethod comprises the steps: i) amplifying PCV2 ORF2 DNA in vitro,wherein the flanking sequences of said PCV2 ORF2 DNA are modified, ii)cloning the amplified PCV2 ORF2 DNA into a transfer vector; and iii)transfecting the transfer vector or a portion thereof containing therecombinant PCV2 ORF2 DNA into a viral vector to generate therecombinant viral vector, iv) infecting cells in media with thetransfected virus; v) causing the transfected virus to express therecombinant protein from PCV2 ORF2; vi) separating cells from thesupernate; vii) recovering the expressed PCV2 ORF2 protein from thesupernate; viii) inactivating the recombinant viral vector, preferably,in the presence of about 1 to about 20 mM BEI, most preferred in thepresence of about 5 mM BEI; and ix) adding of an equivalent amount of anagent that neutralizes the inactivation agent within the solution,preferably, adding of a sodium thiosulfate solution to a finalconcentration of about 1 to about 20 mM, preferably of about 5 mM, whenthe inactivation agent is BEI.

In another aspect of the present invention, a method for preparing acomposition, preferably an antigenic composition, such as for example avaccine, for invoking an immune response against PCV2 is provided.Generally, this method includes the steps of transfecting a constructinto a virus, wherein the construct comprises i) recombinant DNA fromORF2 of PCV2, ii) infecting cells in growth media with the transfectedvirus, iii) causing the virus to express the recombinant protein fromPCV2 ORF2, iv) recovering the expressed ORF2 protein from the supernate,v) and preparing the composition by combining the recovered protein witha suitable adjuvant and/or other pharmaceutically acceptable carrier.

“Adjuvants” as used herein, can include aluminum hydroxide and aluminumphosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc.,Cambridge Mass.), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham,Ala.), water-in-oil emulsion, oil-in-water emulsion,water-in-oil-in-water emulsion. The emulsion can be based in particularon light liquid paraffin oil (European Pharmacopea type); isoprenoid oilsuch as squalane or squalene; oil resulting from theoligomerization ofalkenes, in particular of isobutene or decene; esters of acids or ofalcohols containing a linear alkyl group, more particularly plant oils,ethyl oleate, propylene glycol di-(caprylate/caprate), glyceryltri-(caprylate/caprate) or propylene glycol dioleate; esters of branchedfatty acids or alcohols, in particular isostearic acid esters. The oilis used in combination with emulsifiers to form the emulsion. Theemulsifiers are preferably nonionic surfactants, in particular esters ofsorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, ofpolyglycerol, of propylene glycol and of oleic, isostearic, ricinoleicor hydroxystearic acid, which are optionally ethoxylated, andpolyoxypropylene-polyoxyethylene copolymer blocks, in particular thePluronic products, especially L121. See Hunter et al., The Theory andPractical Application of Adjuvants (Ed. Stewart-Tull, D. E. S.).JohnWiley and Sons, NY, pp 51-94 (1995) and Todd et al., Vaccine15:564-570 (1997).

For example, it is possible to use the SPT emulsion described on page147 of “Vaccine Design, The Subunit and Adjuvant Approach” edited by M.Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59described on page 183 of this same book.

A further instance of an adjuvant is a compound chosen from the polymersof acrylic or methacrylic acid and the copolymers of maleic anhydrideand alkenyl derivative. Advantageous adjuvant compounds are the polymersof acrylic or methacrylic acid which are cross-linked, especially withpolyalkenyl ethers of sugars or polyalcohols. These compounds are knownby the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Personsskilled in the art can also refer to U.S. Pat. No. 2,909,462 whichdescribes such acrylic polymers cross-linked with a polyhydroxylatedcompound having at least 3 hydroxyl groups, preferably not more than 8,the hydrogen atoms of at least three hydroxyls being replaced byunsaturated aliphatic radicals having at least 2 carbon atoms. Thepreferred radicals are those containing from 2 to 4 carbon atoms, e.g.vinyls, alkyls and other ethylenically unsaturated groups. Theunsaturated radicals may themselves contain other substituents, such asmethyl. The products sold under the name Carbopol; (BF Goodrich, Ohio,USA) are particularly appropriate. They are cross-linked with an allylsucrose or with allyl pentaerythritol. Among then, there may bementioned Carbopol 974P, 934P and 971P. Most preferred is the use ofCabopol 971P. Among the copolymers of maleic anhydride and alkenylderivative, the copolymers EMA (Monsanto) which are copolymers of maleicanhydride and ethylene. The dissolution of these polymers in water leadsto an acid solution that will be neutralized, preferably tophysiological pH, in order to give the adjuvant solution into which theimmunogenic, immunological or vaccine composition itself will beincorporated.

Further suitable adjuvants include, but are not limited to, the RIBIadjuvant system (Ribi Inc.), Block co-polymer (CytRx, Atlanta Ga.),SAF-M (Chiron, Emeryville Calif.), monophosphoryl lipid A, Avridinelipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinantor otherwise), cholera toxin, IMS 1314 or muramyl dipeptide among manyothers.

Preferably, the adjuvant is added in an amount of about 100 μg to about10 mg per dose. Even more preferred the adjuvant is added in an amountof about 100 μg to about 10 mg per dose. Even more preferred theadjuvant is added in an amount of about 500 μg to about 5 mg per dose.Even more preferred the adjuvant is added in an amount of about 750 μgto about 2.5 mg per dose. Most preferred the adjuvant is added in anamount of about 1 mg per dose.

Thus, according to a further aspect, the method for preparing anantigenic composition, such as for example a vaccine, for invoking animmune response against PCV2 comprises i) preparing and recovering PCV2ORF2 protein, and ii) admixing this with a suitable adjuvant.Preferably, the adjuvant is Carbopol 971P. Even more preferred, Carbopol971P is added in an amount of about 500 μg to about 5 mg per dose, evenmore preferred in an amount of about 750 μg to about 2.5 mg per dose andmost preferred in an amount of about 1 mg per dose. Preferably, theprocess step i) includes the process steps as described for thepreparation and recovery of PCV2 ORF2. For example, in preferred formsof this method, the construct comprising PCV2 ORF2 DNA is obtained in atransfer vector. Suitable transfer vectors and methods of preparing themare described above. Optionally, the method may include the step ofamplifying the ORF2 from a strain of PCV2 through PCR prior to cloningthe ORF2 into the transfer vector. Preferred open reading framesequences, Kozak's sequences, 3′ EcoR1 site sequences, recombinantprotein sequences, transfected construct sequences, media, cells, andviruses are as described in the previous methods. Another optional stepfor this method includes cloning the amplified PCV2 ORF2 DNA into afirst vector, excising the ORF2 DNA from this first vector, and usingthis excised PCV2 ORF2 DNA for cloning into the transfer vector. As withthe other methods, it is preferred to wait for at least 5 days afterinfection of the cells by the transfected baculovirus prior to recoveryof recombinant ORF2 protein from the supernate. Preferably, the recoverystep of this method also includes the step of separating the media fromthe cells and cell debris. This can be done in a variety of ways but forease and convenience, it is preferred to filter the cells, cell debris,and growth media through a filter having pores ranging in size fromabout 0.45 μM to about 1.0 μM. Finally, for this method, it is preferredto include a virus inactivation step prior to combining the recoveredrecombinant PCV2 ORF2 protein in a composition. This can be done in avariety of ways, but it is preferred in the practice of the presentinvention to use BEI.

Thus according to a further aspect, this method comprises the steps: i)amplifying PCV2 ORF2 DNA in vitro, wherein the flanking sequences ofsaid PCV2 ORF2 DNA are modified, ii) cloning the amplified PCV2 ORF2 DNAinto a transfer vector; and iii) transfecting the transfer vector or aportion thereof containing the recombinant PCV2 ORF2 DNA into a viralvector to generate the recombinant viral vector, iv) infecting cells inmedia with the transfected virus; v) causing the transfected virus toexpress the recombinant protein from PCV2 ORF2; vi) separating cellsfrom the supernate; vii) recovering the expressed PCV2 ORF2 protein fromthe supernate; viii) inactivating the recombinant viral vector,preferably, in the presence of about 1 to about 20 mM BEI, mostpreferred in the presence of about 5 mM BEI; ix) adding of an equivalentamount of an agent that neutralizes the inactivation agent within thesolution, preferably, adding of a sodium thiosulfate solution to a finalconcentration of about 1 to about 20 mM, preferably of about 5 mM, whenthe inactivation agent is BEI, and x) adding a suitable amount of anadjuvant, preferably adding Carbopol, more preferably Carbopol 971P,even more preferred in amounts as described above (e.g. of about 500 μgto about 5 mg per dose, even more preferred in an amount of about 750 μgto about 2.5 mg per dose and most preferred in an amount of about 1 mgper dose).

Additionally, the composition can include one or morepharmaceutical-acceptable carriers. As used herein, “apharmaceutical-acceptable carrier” includes any and all solvents,dispersion media, coatings, stabilizing agents, diluents, preservatives,antibacterial and antifungal agents, isotonic agents, adsorptiondelaying agents, and the like. Most preferred, the composition providedherewith, contains PCV2 ORF2 protein recovered from the supernate of invitro cultured cells, wherein said cells were infected with arecombinant viral vector containing PCV2 ORF2 DNA and expressing PCV2ORF2 protein, and wherein said cell culture were treated with about 2 toabout 8 mM BEI, preferably with about 5 mM BEI to inactivate the viralvector, and an equivalent concentration of a neutralization agent,preferably sodium thiosulfate solution to a final concentration of about2 to about 8 mM, preferably of about 5 mM, Carbopol, more preferablyCarbopol 971P, preferably in amounts of about 500 μg to about 5 mg perdose, even more preferred in an amount of about 750 μg to about 2.5 mgper dose and most preferred in an amount of about 1 mg per dose, andphysiological saline, preferably in an amount of about 50 to about 90%(v/v), more preferably to about 60 to 80% (v/v), still more preferablyof about 70% (v/v).

Thus, a further aspect relates to a method for preparing an antigeniccomposition, such as for example a vaccine, for invoking an immuneresponse against PCV2 comprising the steps: i) amplifying PCV2 ORF2 DNAin vitro, wherein the flanking sequences of said PCV2 ORF2 DNA aremodified, ii) cloning the amplified PCV2 ORF2 DNA into a transfervector; and iii) transfecting the transfer vector or a portion thereofcontaining the recombinant PCV2 ORF2 DNA into a viral vector to generatethe recombinant viral vector, iv) infecting cells in media with thetransfected virus; v) causing the transfected virus to express therecombinant protein from PCV2 ORF2; vi) separating cells from thesupernate; vii) recovering the expressed PCV2 ORF2 protein from thesupernate; viii) inactivating the recombinant viral vector, preferably,in the presence of about 2 to about 20 mM BEI, most preferred in thepresence of about 5 mM BEI; ix) adding of an equivalent amount of anagent that neutralize the inactivation agent within the solution,preferably, adding of a sodium thiosulfate solution to a finalconcentration of about 0.5 to about 20 mM, preferably of about 5 mM,when the inactivation agent is BEI, x) adding a suitable amount of anadjuvants, preferably adding Carbopol, more preferably Carbopol 971P,still more preferred in amounts as described above (e.g. of about 500 μgto about 5 mg per dose, even more preferred in an amount of about 750 μgto about 2.5 mg per dose and most preferred in an amount of about 1 mgper dose); and xi) adding physiological saline, preferably in an amountof about 50 to about 90% (v/v), more preferably to about 60 to 80%(v/v), still more preferably of about 70% (v/v). Optionally, this methodcan also include the addition of a protectant. A protectant as usedherein, refers to an anti-microbiological active agent, such as forexample Gentamycin, Merthiolate, and the like. In particular adding of aprotectant is most preferred for the preparation of a multi-dosecomposition. Those anti-microbiological active agents are added inconcentrations effective to prevent the composition of interest for anymicrobiological contamination or for inhibition of any microbiologicalgrowth within the composition of interest.

Moreover, this method can also comprise addition of any stabilizingagent, such as for example saccharides, trehalose, mannitol, saccharoseand the like, to increase and/or maintain product shelf-life. However,it has been surprisingly found, that the resulting formulation isimmunologically effective over a period of at least 24 months, withoutadding any further stabilizing agent.

A further aspect of the present invention relates to the products resultfrom the methods as described above. In particular, the presentinvention relates to a composition of matter comprises recombinantlyexpressed PCV2 ORF2 protein. Moreover, the present invention alsorelates to a composition of matter that comprises recombinantlyexpressed PCV2 ORF2 protein, recovered from the supernate of an insectcell culture. Moreover, the present invention also relates to acomposition of matter comprises recombinantly expressed PCV2 ORF2protein, recovered from the supernate of an insect cell culture.Preferably, this composition of matter also comprises an agent suitablefor the inactivation of viral vectors. Preferably, said inactivationagent is BEI. Moreover, the present invention also relates to acomposition of matter that comprises recombinantly expressed PCV2 ORF2protein, recovered from the supernate of an insect cell culture, andcomprises an agent, suitable for the inactivation of viral vectors,preferably BEI and a neutralization agent for neutralization of theinactivation agent. Preferably, that neutralization agent is sodiumthiosulfate, when BEI is used as an inactivation agent.

In yet another aspect of the present invention, an immunogeniccomposition that induces an immune response and, more preferably,confers protective immunity against the clinical signs of PCV2infection, is provided. The composition generally comprises thepolypeptide, or a fragment thereof, expressed by Open Reading Frame 2(ORF2) of PCV2, as the antigenic component of the composition.

PCV2 ORF2 DNA and protein, as used herein for the preparation of thecompositions and also as used within the processes provided herein is ahighly conserved domain within PCV2 isolates and thereby, any PCV2 ORF2would be effective as the source of the PCV ORF2 DNA and/or polypeptideas used herein. A preferred PCV2 ORF2 protein is that of SEQ ID NO. 11.A preferred PCV ORF2 polypeptide is provided herein as SEQ ID NO. 5, butit is understood by those of skill in the art that this sequence couldvary by as much as 6-10% in sequence homology and still retain theantigenic characteristics that render it useful in immunogeniccompositions. The antigenic characteristics of an immunologicalcomposition can be, for example, estimated by the challenge experimentas provided by Example 4. Moreover, the antigenic characteristic of anmodified antigen is still retained, when the modified antigen confers atleast 70%, preferably 80%, more preferably 90% of the protectiveimmunity as compared to the PCV2 ORF 2 protein, encoded by thepolynucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4. An “immunogeniccomposition” as used herein, means a PCV2 ORF2 protein which elicits an“immunological response” in the host of a cellular and/orantibody-mediated immune response to PCV2 ORF2 protein. Preferably, thisimmunogenic composition is capable to confer protective immunity againstPCV2 infection and the clinical signs associated therewith. In someforms, immunogenic portions of PCV2 ORF2 protein are used as theantigenic component in the composition. The term “immunogenic portion”as used herein refers to truncated and/or substituted forms, orfragments of PCV2 ORF2 protein and/or polynucleotide, respectively.Preferably, such truncated and/or substituted forms, or fragments willcomprise at least 6 contiguous amino acids from the full-length ORF2polypeptide. More preferably, the truncated or substituted forms, orfragments will have at least 10, more preferably at least 15, and stillmore preferably at least 19 contiguous amino acids from the full-lengthORF2 polypeptide. Two preferred sequences in this respect are providedherein as SEQ ID NOs. 9 and 10. It is further understood that suchsequences may be a part of larger fragments or truncated forms. Afurther preferred PCV2 ORF2 polypeptide provided herein is encoded bythe nucleotide sequences of SEQ ID NO: 3 or SEQ ID NO: 4. But it isunderstood by those of skill in the art that this sequence could vary byas much as 6-20% in sequence homology and still retain the antigeniccharacteristics that render it useful in immunogenic compositions. Insome forms, a truncated or substituted form, or fragment of ORF2 is usedas the antigenic component in the composition. Preferably, suchtruncated or substituted forms, or fragments will comprise at least 18contiguous nucleotides from the full-length ORF2 nucleotide sequence,e.g. of SEQ ID NO: 3 or SEQ ID NO: 4. More preferably, the truncated orsubstituted forms, or fragments will have at least 30, more preferablyat least 45, and still more preferably at least 57 contiguousnucleotides the full-length ORF2 nucleotide sequence, e.g. of SEQ ID NO:3 or SEQ ID NO: 4.

“Sequence Identity” as it is known in the art refers to a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, namely a reference sequence and a given sequence to becompared with the reference sequence. Sequence identity is determined bycomparing the given sequence to the reference sequence after thesequences have been optimally aligned to produce the highest degree ofsequence similarity, as determined by the match between strings of suchsequences. Upon such alignment, sequence identity is ascertained on aposition-by-position basis, e.g., the sequences are “identical” at aparticular position if at that position, the nucleotides or amino acidresidues are identical. The total number of such position identities isthen divided by the total number of nucleotides or residues in thereference sequence to give % sequence identity. Sequence identity can bereadily calculated by known methods, including but not limited to, thosedescribed in Computational Molecular Biology, Lesk, A. N., ed., OxfordUniversity Press, New York (1988), Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York (1993); ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey (1994); Sequence Analysis in MolecularBiology, von Heinge, G., Academic Press (1987); Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York(1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988), the teachings of which are incorporated herein by reference.Preferred methods to determine the sequence identity are designed togive the largest match between the sequences tested. Methods todetermine sequence identity are codified in publicly available computerprograms which determine sequence identity between given sequences.Examples of such programs include, but are not limited to, the GCGprogram package (Devereux, J., et al., Nucleic Acids Research, 12(1):387(1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec.Biol., 215:403-410 (1990). The BLASTX program is publicly available fromNCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIHBethesda, Md. 20894, Altschul, S. F. et al., J. Molec. Biol.,215:403-410 (1990), the teachings of which are incorporated herein byreference). These programs optimally align sequences using default gapweights in order to produce the highest level of sequence identitybetween the given and reference sequences. As an illustration, by apolynucleotide having a nucleotide sequence having at least, forexample, 85%, preferably 90%, even more preferably 95% “sequenceidentity” to a reference nucleotide sequence, it is intended that thenucleotide sequence of the given polynucleotide is identical to thereference sequence except that the given polynucleotide sequence mayinclude up to 15, preferably up to 10, even more preferably up to 5point mutations per each 100 nucleotides of the reference nucleotidesequence. In other words, in a polynucleotide having a nucleotidesequence having at least 85%, preferably 90%, even more preferably 95%identity relative to the reference nucleotide sequence, up to 15%,preferably 10%, even more preferably 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 15%, preferably 10%, evenmore preferably 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Analogously, by a polypeptide having a given aminoacid sequence having at least, for example, 85%, preferably 90%, evenmore preferably 95% sequence identity to a reference amino acidsequence, it is intended that the given amino acid sequence of thepolypeptide is identical to the reference sequence except that the givenpolypeptide sequence may include up to 15, preferably up to 10, evenmore preferably up to 5 amino acid alterations per each 100 amino acidsof the reference amino acid sequence. In other words, to obtain a givenpolypeptide sequence having at least 85%, preferably 90%, even morepreferably 95% sequence identity with a reference amino acid sequence,up to 15%, preferably up to 10%, even more preferably up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to15%, preferably up to 10%, even more preferably up to 5% of the totalnumber of amino acid residues in the reference sequence may be insertedinto the reference sequence. These alterations of the reference sequencemay occur at the amino or the carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in the one or more contiguous groups within thereference sequence. Preferably, residue positions which are notidentical differ by conservative amino acid substitutions. However,conservative substitutions are not included as a match when determiningsequence identity.

“Sequence homology”, as used herein, refers to a method of determiningthe relatedness of two sequences. To determine sequence homology, two ormore sequences are optimally aligned, and gaps are introduced ifnecessary. However, in contrast to “sequence identity”, conservativeamino acid substitutions are counted as a match when determiningsequence homology. In other words, to obtain a polypeptide orpolynucleotide having 95% sequence homology with a reference sequence,85%, preferably 90%, even more preferably 95% of the amino acid residuesor nucleotides in the reference sequence must match or comprise aconservative substitution with another amino acid or nucleotide, or anumber of amino acids or nucleotides up to 15%, preferably up to 10%,even more preferably up to 5% of the total amino acid residues ornucleotides, not including conservative substitutions, in the referencesequence may be inserted into the reference sequence. Preferably thehomolog sequence comprises at least a stretch of 50, even more preferredof 100, even more preferred of 250, even more preferred of 500nucleotides.

A “conservative substitution” refers to the substitution of an aminoacid residue or nucleotide with another amino acid residue or nucleotidehaving similar characteristics or properties including size,hydrophobicity, etc., such that the overall functionality does notchange significantly.

Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide orpolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein.

Thus, a further aspect of the present invention relates to animmunogenic composition effective for lessening the severity of clinicalsymptoms associated with PCV2 infection comprising PCV2 ORF2 protein.Preferably, the PCV2 ORF2 protein is anyone of those, described above.Preferably, said PCV2 ORF2 protein is

-   -   i) a polypeptide comprising the sequence of SEQ ID NO: 5, SEQ ID        NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11;    -   ii) any polypeptide that is at least 80% homologous to the        polypeptide of i),    -   iii) any immunogenic portion of the polypeptides of i) and/or        ii)    -   iv) the immunogenic portion of iii), comprising at least 10        contiguous amino acids included in the sequences of SEQ ID NO:        5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11,    -   v) a polypeptide that is encoded by a DNA comprising the        sequence of SEQ ID NO: 3 or SEQ ID NO: 4.    -   vi) any polypeptide that is encoded by a polynucleotide that is        at least 80% homolog to the polynucleotide of v),    -   vii) any immunogenic portion of the polypeptides encoded by the        polynucleotide of v) and/or vi)    -   viii) the immunogenic portion of vii), wherein polynucleotide        coding for said immunogenic portion comprises at least 30        contiguous nucleotides included in the sequences of SEQ ID NO:        3, or SEQ ID NO: 4.

Preferably any of those immunogenic portions having the immunogeniccharacteristics of PCV2 ORF2 protein that is encoded by the sequence ofSEQ ID NO: 3 or SEQ ID NO: 4.

According to a further aspect, PCV2 ORF2 protein is provided in theimmunological composition at an antigen inclusion level effective forinducing the desired immune response, namely reducing the incidence ofor lessening the severity of clinical signs resulting from PCV2infection. Preferably, the PCV2 ORF2 protein inclusion level is at least0.2 μg antigen/ml of the final immunogenic composition (μg/ml), morepreferably from about 0.2 to about 400 μg/ml, still more preferably fromabout 0.3 to about 200 μg/ml, even more preferably from about 0.35 toabout 100 μg/ml, still more preferably from about 0.4 to about 50 μg/ml,still more preferably from about 0.45 to about 30 μg/ml, still morepreferably from about 0.6 to about 15 μg/ml, even more preferably fromabout 0.75 to about 8 μg/ml, even more preferably from about 1.0 toabout 6 μg/ml, still more preferably from about 1.3 to about 3.0 μg/ml,even more preferably from about 1.4 to about 2.5 μg/ml, even morepreferably from about 1.5 to about 2.0 μg/ml, and most preferably about1.6 μg/ml.

According to a further aspect, the ORF2 antigen inclusion level is atleast 0.2 μg PCV2 ORF2 protein as described above per dose of the finalantigenic composition (μg/dose), more preferably from about 0.2 to about400 μg/dose, still more preferably from about 0.3 to about 200 μg/dose,even more preferably from about 0.35 to about 100 μg/dose, still morepreferably from about 0.4 to about 50 μg/dose, still more preferablyfrom about 0.45 to about 30 μg/dose, still more preferably from about0.6 to about 15 μg/dose, even more preferably from about 0.75 to about 8μg/dose, even more preferably from about 1.0 to about 6 μg/dose, stillmore preferably from about 1.3 to about 3.0 μg/dose, even morepreferably from about 1.4 to about 2.5 μg/dose, even more preferablyfrom about 1.5 to about 2.0 μg/dose, and most preferably about 1.6μg/dose.

The PCV2 ORF2 polypeptide used in an immunogenic composition inaccordance with the present invention can be derived in any fashionincluding isolation and purification of PCV2 ORF2, standard proteinsynthesis, and recombinant methodology. Preferred methods for obtainingPCV2 ORF2 polypeptide are described herein above and are also providedin U.S. patent application Ser. No. 11/034,797, the teachings andcontent of which are hereby incorporated by reference. Briefly,susceptible cells are infected with a recombinant viral vectorcontaining PCV2 ORF2 DNA coding sequences, PCV2 ORF2 polypeptide isexpressed by the recombinant virus, and the expressed PCV2 ORF2polypeptide is recovered from the supernate by filtration andinactivated by any conventional method, preferably using binaryethylenimine, which is then neutralized to stop the inactivationprocess.

Thus, according to a further aspect the immunogenic compositioncomprises i) any of the PCV2 ORF2 protein described above, preferably inconcentrations described above, and ii) at least a portion of the viralvector expressing said PCV2 ORF2 protein, preferably of a recombinantbaculovirus. Moreover, according to a further aspect, the immunogeniccomposition comprises i) any of the PCV2 ORF2 protein described above,preferably in concentrations described above, ii) at least a portion ofthe viral vector expressing said PCV2 ORF2 protein, preferably of arecombinant baculovirus, and iii) a portion of the cell culturesupernate.

According to one specific embodiment of the production and recoveryprocess for PCV2 ORF2 protein, the cell culture supernate is filteredthrough a membrane having a pore size, preferably between about 0.45 to1 μm. Thus, a further aspect relates to an immunogenic composition thatcomprises i) any of the PCV2 ORF2 protein described above, preferably inconcentrations described above, ii) at least a portion of the viralvector expressing said PCV2 ORF2 protein, preferably of a recombinantbaculovirus, and iii) a portion of the cell culture; wherein about 90%of the components have a size smaller than 1 μm.

According to a further aspect, the present invention relates to animmunogenic composition that comprises i) any of the PCV2 ORF2 proteindescribed above, preferably in concentrations described above, ii) atleast a portion of the viral vector expressing said PCV2 ORF2 protein,iii) a portion of the cell culture, iv) and inactivating agent toinactivate the recombinant viral vector preferably BEI, wherein about90% of the components i) to iii) have a size smaller than 1 μm.Preferably, BEI is present in concentrations effective to inactivate thebaculovirus. Effective concentrations are described above.

According to a further aspect, the present invention relates to animmunogenic composition that comprises i) any of the PCV2 ORF2 proteindescribed above, preferably in concentrations described above, ii) atleast a portion of the viral vector expressing said PCV2 ORF2 protein,iii) a portion of the cell culture, iv) an inactivating agent toinactivate the recombinant viral vector preferably BEI, and v) anneutralization agent to stop the inactivation mediated by theinactivating agent, wherein about 90% of the components i) to iii) havea size smaller than 1 μm. Preferably, if the inactivating agent is BEI,said composition comprises sodium thiosulfate in equivalent amounts toBEI.

The polypeptide is incorporated into a composition that can beadministered to an animal susceptible to PCV2 infection. In preferredforms, the composition may also include additional components known tothose of skill in the art (see also Remington's Pharmaceutical Sciences.(1990). 18th ed. Mack Publ., Easton). Additionally, the composition mayinclude one or more veterinary-acceptable carriers. As used herein, “aveterinary-acceptable carrier” includes any and all solvents, dispersionmedia, coatings, adjuvants, stabilizing agents, diluents, preservatives,antibacterial and antifungal agents, isotonic agents, adsorptiondelaying agents, and the like.

In a preferred embodiment, the immunogenic composition comprises PCV2ORF2 protein as provided herewith, preferably in concentrationsdescribed above as an antigenic component, which is mixed with anadjuvant, preferably Carbopol, and physiological saline.

Those of skill in the art will understand that the composition hereinmay incorporate known injectable, physiologically acceptable sterilesolutions. For preparing a ready-to-use solution for parenteralinjection or infusion, aqueous isotonic solutions, such as e.g. salineor corresponding plasma protein solutions are readily available. Inaddition, the immunogenic and vaccine compositions of the presentinvention can include diluents, isotonic agents, stabilizers, oradjuvants. Diluents can include water, saline, dextrose, ethanol,glycerol, and the like. Isotonic agents can include sodium chloride,dextrose, mannitol, sorbitol, and lactose, among others. Stabilizersinclude albumin and alkali salts of ethylendiamintetracetic acid, amongothers. Suitable adjuvants, are those described above. Most preferred isthe use of Carbopol, in particular the use of Carbopol 971P, preferablyin amounts as described above (e.g. of about 500 μg to about 5 mg perdose, even more preferred in an amount of about 750 μg to about 2.5 mgper dose and most preferred in an amount of about 1 mg per dose).

Thus, the present invention also relates to an immunogenic compositionthat comprises i) any of the PCV2 ORF2 proteins described above,preferably in concentrations described above, ii) at least a portion ofthe viral vector expressing said PCV2 ORF2 protein, iii) a portion ofthe cell culture, iv) an inactivating agent to inactivate therecombinant viral vector preferably BEI, and v) an neutralization agentto stop the inactivation mediated by the inactivating agent, preferablysodium thiosulfate in equivalent amounts to BEI; and vi) a suitableadjuvant, preferably Carbopol 971 in amounts described above; whereinabout 90% of the components i) to iii) have a size smaller than 1 μm.According to a further aspect, this immunogenic composition furthercomprises a pharmaceutical acceptable salt, preferably a phosphate saltin physiologically acceptable concentrations. Preferably, the pH of saidimmunogenic composition is adjusted to a physiological pH, meaningbetween about 6.5 and 7.5.

Thus, the present invention also relates to an immunogenic compositioncomprises per one ml i) at least 1.6 μg of PCV2 ORF2 protein describedabove, ii) at least a portion of baculovirus expressing said PCV2 ORF2protein iii) a portion of the cell culture, iv) about 2 to 8 mM BEI, v)sodium thiosulfate in equivalent amounts to BEI; and vi) about 1 mgCarbopol 971, and vii) phosphate salt in a physiologically acceptableconcentration; wherein about 90% of the components i) to iii) have asize smaller than 1 μm and the pH of said immunogenic composition isadjusted to about 6.5 to 7.5.

The immunogenic compositions can further include one or more otherimmunomodulatory agents such as, e.g., interleukins, interferons, orother cytokines. The immunogenic compositions can also includeGentamicin and Merthiolate. While the amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan, the present inventioncontemplates compositions comprising from about 50 μg to about 2000 μgof adjuvant and preferably about 250 μg/ml dose of the vaccinecomposition. In another preferred embodiment, the present inventioncontemplates vaccine compositions comprising from about 1 ug/ml to about60 μg/ml of antibiotics, and more preferably less than about 30 μg/ml ofantibiotics.

Thus, the present invention also relates to an immunogenic compositionthat comprises i) any of the PCV2 ORF2 proteins described above,preferably in concentrations described above, ii) at least a portion ofthe viral vector expressing said PCV2 ORF2 protein, iii) a portion ofthe cell culture, iv) an inactivating agent to inactivate therecombinant viral vector preferably BEI, and v) an neutralization agentto stop the inactivation mediated by the inactivating agent, preferablysodium thiosulfate in equivalent amounts to BEI; vi) a suitableadjuvant, preferably Carbopol 971 in amounts described above; vii) apharmaceutical acceptable concentration of a saline buffer, preferablyof a phosphate salt, and viii) an anti-microbiological active agent;wherein about 90% of the components i) to have a size smaller than 1 μm.

It has been surprisingly found, that the immunogenic compositionprovided herewith comprises was highly stable over a period of 24months. It has also been found the immunogenic compositions providedherewith, comprising recombinant, baculovirus expressed PCV2 ORF2protein as provided herewith are very effective in reducing the clinicalsymptoms associated with PCV2 infections. It has been surprisinglyfound, that the immunogenic compositions comprising the recombinantbaculovirus expressed PCV2 ORF2 protein as provided herewith, are moreeffective than an immunogenic composition comprising the whole PCV2virus in an inactivated form, or isolated viral PCV2 ORF2 antigen. Inparticular, it has been surprisingly found, that the recombinantbaculovirus expressed PCV2 ORF2 protein is effective is in very lowconcentrations, which means in concentrations up to 0.25 μg/dose. Thisunexpected high immunogenic potential of the PCV2 ORF2 protein could befurther increased by the addition of Carbopol.

A further aspect relates to a container comprises at least one dose ofthe immunogenic composition of PCV2 ORF2 protein as provided herewith,wherein one dose comprises at least 2 μg PCV2 ORF2 protein, preferably 2to 16 μg PCV2 ORF2 protein. Said container can comprises 1 to 250 dosesof the immunogenic composition, preferably it contains 1, 10, 25, 50,100, 150, 200, or 250 doses of the immunogenic composition of PCV2 ORF2protein. Preferably, each of the containers comprising more than onedose of the immunogenic composition of PCV2 ORF2 protein furthercomprises an anti-microbiological active agent. Those agents are for forexample antibiotics including Gentamicin and Merthiolate and the like.Thus, one aspect of the present invention relates to a container thatcomprises 1 to 250 doses of the immunogenic composition of PCV2 ORF2protein, wherein one dose comprises at least 2 μg PCV2 ORF2 protein, andGentamicin and/or Merthiolate, preferably from about 1 μg/ml to about 60μg/ml of antibiotics, and more preferably less than about 30 μg/ml.

A further aspect relates to a kit, comprising any of the containers,described above, and an instruction manual, including the informationfor the intramuscular application of at least one dose of theimmunogenic composition of PCV2 ORF2 protein into piglets to lesseningthe severity of clinical symptoms associated with PCV2 infection.Moreover, according to a further aspect, said instruction manualcomprises the information of a second or further administration(s) of atleast one dose of the immunogenic composition of PCV2 ORF2, wherein thesecond administration or any further administration is at least 14 daysbeyond the initial or any former administration. Preferably, saidinstruction manual also includes the information, to administer animmune stimulant. Preferably, said immune stimulant shall be given atleast twice. Preferably, at least 3, more preferably at least 5, evenmore preferably at least 7 days are between the first and the second orany further administration of the immune stimulant. Preferably, theimmune stimulant is given at least 10 days, preferably 15, even morepreferably 20, even more preferably at least 22 days beyond the initialadministration of the immunogenic composition of PCV2 ORF2 protein. Apreferred immune stimulant is for example is keyhole limpet hemocyanin(KLH), still preferably emulsified with incomplete Freund's adjuvant(KLH/ICFA). However, it is herewith understood, that any other immunestimulant known to a person skilled in the art can also be used “Immunestimulant” as used herein, means any agent or composition that cantrigger the immune response, preferably without initiating or increasinga specific immune response, for example the immune response against aspecific pathogen. It is further instructed to administer the immunestimulant in a suitable dose. Moreover, the kit may also comprises acontainer, including at least one dose of the immune stimulant,preferably one dose of KLH, or KLH/ICFA.

Moreover, it has also been surprisingly found that the immunogenicpotential of the immunogenic compositions comprising recombinantbaculovirus expressed PCV2 ORF2 protein, preferably in combination withCarbopol, can be further enhanced by the administration of the IngelVacPRRS MLV vaccine (see Example 5). PCV2 clinical signs and diseasemanifestations are greatly magnified when PRRS infection is present.However, the immunogenic compositions and vaccination strategies asprovided herewith lessened this effect greatly, and more than expected.In other words, an unexpected synergistic effect was observed whenanimals, preferably pigs are treated with any of the PCV2 ORF2immunogenic composition, as provided herewith, and the Ingelvac PRRS MLVvaccine (Boehringer Ingelheim).

Thus, a further aspect of the present invention relates to the kit asdescribed above, comprising the immunogenic composition of PCV2 ORF2 asprovided herewith and the instruction manual, wherein the instructionmanual further include the information to administer the PCV2 ORF2immunogenic composition together with immunogenic composition thatcomprises PRRS antigen, preferably adjuvanted PRRS antigen. Preferably,the PRRS antigen is adjuvanted with Carbopol. Preferably, the PRRSantigen is IngelVac® PRRS MLV (Boehringer Ingelheim).

A further aspect of the present invention also relates to a kitcomprising i) a container containing at least one dose of an immunogeniccomposition of PCV2 ORF2 as provided herewith, and ii) a containercontaining an immunogenic composition comprising PRRS antigen,preferably adjuvanted PRRS antigen. Preferably, the PRRS antigen isadjuvanted with Carbopol. Preferably the PRRS antigen is IngelVac® PRRSMLV (Boehringer Ingelheim). More preferably, the kit further comprisesan instruction manual, including the information to administer bothpharmaceutical compositions. Preferably, it contains the informationthat the PCV2 ORF2 containing composition is administered temporallyprior to the PRRS containing composition.

A further aspect, relates to the use of any of the compositions providedherewith as a medicament, preferably as a veterinary medicament, evenmore preferred as a vaccine. Moreover, the present invention alsorelates to the use of any of the compositions described herein, for thepreparation of a medicament for lessening the severity of clinicalsymptoms associated with PCV2 infection. Preferably, the medicament isfor the prevention of a PCV2 infection, even more preferably in piglets.

A further aspect relates to a method for (i) the prevention of aninfection, or re-infection with PCV2 or (ii) the reduction orelimination of clinical symptoms caused by PCV2 in a subject, comprisingadministering any of the immunogenic compositions provided herewith to asubject in need thereof. Preferably, the subject is a pig. Preferably,the immunogenic composition is administered intramuscular. Preferably,one dose or two doses of the immunogenic composition is/areadministered, wherein one dose preferably comprises at least about 2 μgPCV2 ORF2 protein, even more preferably about 2 to about 16 μg, and atleast about 0.1 to about 5 mg Carbopol, preferably about 1 mg Carbopol.A further aspect relates to the method of treatment as described above,wherein a second application of the immunogenic composition isadministered. Preferably, the second administration is done with thesame immunogenic composition, preferably having the same amount of PCV2ORF2 protein. Preferably the second administration is also givenintramuscular. Preferably, the second administration is done at least 14days beyond the initial administration, even more preferably at least 4weeks beyond the initial administration.

According to a further aspect, the method of treatment also comprisesthe administration of an immune stimulant. Preferably, said immunestimulant is administered at least twice. Preferably, at least 3, morepreferably at least 5 days, even more preferably at least 7 days arebetween the first and the second administration of the immune stimulant.Preferably, the immune stimulant is administered at least 10 days,preferably 15, even more preferably 20, even more preferably at least 22days beyond the initial administration of the PCV2 ORF2 immunogeniccomposition. A preferred immune stimulant is for example is keyholelimpet hemocyanin (KLH), still preferably emulsified with incompleteFreund's adjuvant (KLH/ICFA). However, it is herewith understood, thatany other immune stimulant known to a person skilled in the art can alsobe used. It is within the general knowledge of a person skilled in theart to administer the immune stimulant in a suitable dose.

According to a further aspect, the method of treatments described abovealso comprises the administration of PRRS antigen. Preferably, the PRRSantigen is adjuvanted with Carbopol. Preferably the PRRS antigen isIngelVac® PRRS MLV (Boehringer Ingelheim). Preferably, said PRRS antigenis administered temporally beyond the administration of the immunogeniccomposition of PCV2 ORF2 protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic flow diagram of a preferred construction of PCV2ORF2 recombinant baculovirus; and

FIGS. 2 a and 2 b are a schematic flow diagram of how to produce acomposition in accordance with the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples set forth preferred materials and procedures inaccordance with the present invention. It is to be understood, however,that these examples are provided by way of illustration only, andnothing therein should be deemed a limitation upon the overall scope ofthe invention.

Example 1

This example compares the relative yields of ORF2 using methods of thepresent invention with methods that are known in the prior art. Four1000 mL spinner flasks were each seeded with approximately 1.0×10⁶ Sf+cells/ml in 300 mL of insect serum free media, Excel 420 (JRHBiosciences, Inc., Lenexa, Kans.). The master cell culture is identifiedas SF+ (Spodoptera frugiperda) Master Cell Stock, passage 19,Lot#N112-095W. The cells used to generate the SF+ Master Cell Stock wereobtained from Protein Sciences Corporation, Inc., Meriden, Conn. The SF+cell line for this example was confined between passages 19 and 59.Other passages will work for purposes of the present invention, but inorder to scale the process up for large scale production, at least 19passages will probably be necessary and passages beyond 59 may have aneffect on expression, although this was not investigated. In moredetail, the initial SF+ cell cultures from liquid nitrogen storage weregrown in Excel 420 media in suspension in sterile spinner flasks withconstant agitation. The cultures were grown in 100 mL to 250 mL spinnerflasks with 25 to 150 mL of Excel 420 serum-free media. When the cellshad multiplied to a cell density of 1.0-8.0×10⁶ cells/mL, they weresplit to new vessels with a planting density of 0.5-1.5×10⁶ cells/mL.Subsequent expansion cultures were grown in spinner flasks up to 36liters in size or in stainless steel bioreactors of up to 300 liters fora period of 2-7 days at 25-29° C.

After seeding, the flasks were incubated at 27° C. for four hours.Subsequently, each flask was seeded with a recombinant baculoviruscontaining the PCV2 ORF2 gene (SEQ ID NO: 4). The recombinantbaculovirus containing the PCV2 ORF2 gene was generated as follows: thePCV2 ORF2 gene from a North American strain of PCV2 was PCR amplified tocontain a 5′ Kozak's sequence (SEQ ID NO: 1) and a 3′ EcoR1 site (SEQ IDNO: 2), cloned into the pGEM-T-Easy vector (Promega, Madison, Wis.).Then, it was subsequently excised and subcloned into the transfer vectorpVL1392 (BD Biosciences Pharmingen, San Diego, Calif.). The subclonedportion is represented herein as SEQ ID NO: 7. The pVL1392 plasmidcontaining the PCV2 ORF2 gene was designated N47-064Y and thenco-transfected with BaculoGold® (BD Biosciences Pharmingen) baculovirusDNA into Sf+ insect cells (Protein Sciences, Meriden, Conn.) to generatethe recombinant baculovirus containing the PCV2 ORF2 gene. The newconstruct is provided herein as SEQ ID NO: 8. The recombinantbaculovirus containing the PCV2 ORF2 gene was plaque-purified and MasterSeed Virus (MSV) was propagated on the SF+ cell line, aliquoted, andstored at −70° C. The MSV was positively identified as PCV2 ORF2baculovirus by PCR-RFLP using baculovirus specific primers. Insect cellsinfected with PCV2 ORF2 baculovirus to generate MSV or Working SeedVirus express PCV2 ORF2 antigen as detected by polyclonal serum ormonoclonal antibodies in an indirect fluorescent antibody assay.Additionally, the identity of the PCV2 ORF2 baculovirus was confirmed byN-terminal amino acid sequencing. The PCV2 ORF2 baculovirus MSV was alsotested for purity in accordance with 9 C.F.R. 113.27 (c), 113.28, and113.55. Each recombinant baculovirus seeded into the spinner flasks hadvarying multiplicities of infection (MOIs). Flask 1 was seeded with 7.52mL of 0.088 MOI seed; flask 2 was seeded with 3.01 mL of 0.36 MOI seed;flask 3 was seeded with 1.5 mL of 0.18 MOI seed; and flask 4 was seededwith 0.75 mL of 0.09 MOI seed. A schematic flow diagram illustrating thebasic steps used to construct a PCV2 ORF2 recombinant baculovirus isprovided herein as FIG. 1.

After being seeded with the baculovirus, the flasks were then incubatedat 27±2° C. for 7 days and were also agitated at 100 rpm during thattime. The flasks used ventilated caps to allow for air flow. Samplesfrom each flask were taken every 24 hours for the next 7 days. Afterextraction, each sample was centrifuged, and both the pellet and thesupernatant were separated and then microfiltered through a 0.45-1.0 μmpore size membrane.

The resulting samples then had the amount of ORF2 present within themquantified via an ELISA assay. The ELISA assay was conducted withcapture antibody Swine anti-PCV2 Pab IgG Prot. G purified (diluted 1:250in PBS) diluted to 1:6000 in 0.05M Carbonate buffer (pH 9.6). 100 μL ofthe antibody was then placed in the wells of the microtiter plate,sealed, and incubated overnight at 37° C. The plate was then washedthree times with a wash solution which comprised 0.5 mL of Tween 20(Sigma, St. Louis, Mo.), 100 mL of 10× D-PBS (Gibco Invitrogen,Carlsbad, Calif.) and 899.5 mL of distilled water. Subsequently, 250 μLof a blocking solution (5 g Carnation Non-fat dry milk (Nestle,Glendale, Calif.) in 10 mL of D-PBS QS to 100 mL with distilled water)was added to each of the wells. The next step was to wash the test plateand then add pre-diluted antigen. The pre-diluted antigen was producedby adding 200 μL of diluent solution (0.5 mL Tween 20 in 999.5 mL D-PBS)to each of the wells on a dilution plate. The sample was then diluted ata 1:240 ratio and a 1:480 ratio, and 100 μL of each of these dilutedsamples was then added to one of the top wells on the dilution plate(i.e. one top well received 100 μL of the 1:240 dilution and the otherreceived 100 μL of the 1:480 dilution). Serial dilutions were then donefor the remainder of the plate by removing 100 μL form each successivewell and transferring it to the next well on the plate. Each well wasmixed prior to doing the next transfer. The test plate washing includedwashing the plate three times with the wash buffer. The plate was thensealed and incubated for an hour at 37° C. before being washed threemore times with the wash buffer. The detection antibody used wasmonoclonal antibody to PCV ORF2. It was diluted to 1:300 in diluentsolution, and 100 μL of the diluted detection antibody was then added tothe wells. The plate was then sealed and incubated for an hour at 37° C.before being washed three times with the wash buffer. Conjugate diluentwas then prepared by adding normal rabbit serum (Jackson Immunoresearch,West Grove, Pa.) to the diluent solution to 1% concentration. Conjugateantibody Goat anti-mouse (H+1)-HRP (Jackson Immunoresearch) was dilutedin the conjugate diluent to 1:10,000. 100 μL of the diluted conjugateantibody was then added to each of the wells. The plate was then sealedand incubated for 45 minutes at 37° C. before being washed three timeswith the wash buffer. 100 μL of substrate (TMB Peroxidase Substrate,Kirkgaard and Perry Laboratories (KPL), Gaithersberg, Md.), mixed withan equal volume of Peroxidase Substrate B (KPL) was added to each of thewells. The plate was incubated at room temperature for 15 minutes. 100μL of 1N HCL solution was then added to all of the wells to stop thereaction. The plate was then run through an ELISA reader. The results ofthis assay are provided in Table 1 below:

TABLE 1 Day Flask ORF2 in pellet (μg) ORF2 in supernatant (μg) 3 1 47.5312 3 2 57.46 22 3 3 53.44 14 3 4 58.64 12 4 1 43.01 44 4 2 65.61 62 4 370.56 32 4 4 64.97 24 5 1 31.74 100 5 2 34.93 142 5 3 47.84 90 5 4 55.1486 6 1 14.7 158 6 2 18.13 182 6 3 34.78 140 6 4 36.88 146 7 1 6.54 176 72 12.09 190 7 3 15.84 158 7 4 15.19 152

These results indicate that when the incubation time is extended,expression of ORF2 into the supernatant of the centrifuged cells andmedia is greater than expression in the pellet of the centrifuged cellsand media. Accordingly, allowing the ORF2 expression to proceed for atleast 5 days and recovering it in the supernate rather than allowingexpression to proceed for less than 5 days and recovering ORF2 from thecells, provides a great increase in ORF2 yields, and a significantimprovement over prior methods.

Example 2

This example provides data as to the efficacy of the invention claimedherein. A 1000 mL spinner flask was seeded with approximately 1.0×10⁶Sf+cells/ml in 300 mL of Excel 420 media. The flask was then incubated at27° C. and agitated at 100 rpm. Subsequently, the flask was seeded with10 mL of PCV2 ORF2/Bac p+6 (the recombinant baculovirus containing thePCV2 ORF2 gene passaged 6 additional times in the Sf9 insect cells)virus seed with a 0.1 MOI after 24 hours of incubation.

The flask was then incubated at 27° C. for a total of 6 days. Afterincubation, the flask was then centrifuged and three samples of theresulting supernatant were harvested and inactivated. The supernatantwas inactivated by bringing its temperature to 37±2° C. To the firstsample, a 0.4M solution of 2-bromoethyleneamine hydrobromide which hadbeen cyclized to 0.2M binary ethlylenimine (BEI) in 0.3N NaOH is addedto the supernatant to give a final concentration of BEI of 5 mM. To thesecond sample, 10 mM BEI was added to the supernatant. To the thirdsample, no BEI was added to the supernatant. The samples were thenstirred continuously for 48 hrs. A 1.0 M sodium thiosulfate solution togive a final minimum concentration of 5 mM was added to neutralize anyresidual BEI. The quantity of ORF2 in each sample was then quantifiedusing the same ELISA assay procedure as described in Example 1. Theresults of this may be seen in Table 2 below:

TABLE 2 Sample ORF2 in supernatant (μg) 1 78.71 2 68.75 3 83.33

This example demonstrates that neutralization with BEI does not removeor degrade significant amounts of the recombinant PCV2 ORF2 proteinproduct. This is evidenced by the fact that there is no large loss ofORF2 in the supernatant from the BEI or elevated temperatures. Those ofskill in the art will recognize that the recovered ORF2 is a stableprotein product.

Example 3

This example demonstrates that the present invention is scalable fromsmall scale production of recombinant PCV2 ORF2 to large scaleproduction of recombinant PCV2 ORF2. 5.0×10⁵ cells/ml of SF+ cells/ml in7000 mL of ExCell 420 media was planted in a 20000 mL ApplikonBioreactor. The media and cells were then incubated at 27° C. andagitated at 100 RPM for the next 68 hours. At the 68^(th) hour, 41.3 mLof PCV2 ORF2 Baculovirus MSV+3 was added to 7000 mL of ExCell 420medium. The resultant mixture was then added to the bioreactor. For thenext seven days, the mixture was incubated at 27° C. and agitated at 100RPM. Samples from the bioreactor were extracted every 24 hours beginningat day 4, post-infection, and each sample was centrifuged. Thesupernatant of the samples were preserved and the amount of ORF2 wasthen quantified using SDS-PAGE densitometry. The results of this can beseen in Table 3 below:

TABLE 3 Day after infection: ORF2 in supernatant (μg/mL) 4 29.33 5 41.336 31.33 7 60.67

Example 4

This example tests the efficacy of seven PCV2 candidate vaccines andfurther defines efficacy parameters following exposure to a virulentstrain of PCV2. One hundred and eight (108) cesarean derived colostrumdeprived (CDCD) piglets, 9-14 days of age, were randomly divided into 9groups of equal size. Table 4 sets forth the General Study Design forthis Example.

TABLE 4 General Study Design KLH/ICFA Challenged No. Of Day of on Day 21with Virulent Necropsy Group Pigs Treatment Treatment and Day 27 PCV2 onDay 24 on Day 49 1 12 PCV2 Vaccine No. 1 - 0 + + + (vORF2 16 μg) 2 12PCV2 Vaccine No. 2 - 0 + + + (vORF2 8 μg) 3 12 PCV2 Vaccine No. 3 -0 + + + (vORF2 4 μg) 4 12 PCV2 Vaccine No. 4 - 0 + + + (rORF2 16 μg) 512 PCV2 Vaccine No. 5 - 0 + + + (rORF2 8 μg) 6 12 PCV2 Vaccine No. 6 -0 + + + (rORF2 4 μg) 7 12 PCV2 Vaccine No. 7 - 0 + + + (Killed wholecell virus) 8 12 None - Challenge N/A + + + Controls 9 12 None - StrictN/A + − + Negative Control Group vORF2 = isolated viral ORF2; rORF2 =recombinant baculovirus expressed ORF2; killed whole cell virus = PCV2virus grown in suitable cell culture

Seven of the groups (Groups 1-7) received doses of PCV2 ORF2polypeptide, one of the groups acted as a challenge control and receivedno PCV2 ORF2, and another group acted as the strict negative controlgroup and also received no PCV2 ORF2. On Day 0, Groups 1 through 7 weretreated with assigned vaccines. Piglets in Group 7 were given a boostertreatment on Day 14. Piglets were observed for adverse events andinjection site reactions following vaccination and on Day 19, pigletswere moved to the second study site. At the second study site, Groups1-8 were group housed in one building while Group 9 was housed in aseparate building. All pigs received keyhole limpet hemocyanin(KLH)/incomplete Freund's adjuvant (ICFA) on Days 21 and 27 and on Day24, Groups 1-8 were challenged with a virulent PCV2.

Pre- and post-challenge, blood samples were collected for PCV2 serology.Post-challenge, body weight data for determination of average dailyweight gain (ADWG), and clinical symptoms, as well as nasal swab samplesto determine nasal shedding of PCV2, were collected. On Day 49, allsurviving pigs were necropsied, lungs were scored for lesions, andselected tissues were preserved in formalin for Immunohistochemistry(IHC) testing at a later date.

Materials and Methods

This was a partially blinded vaccination-challenge feasibility studyconducted in CDCD pigs, 9 to 14 days of age on Day 0. To be included inthe study, PCV2 IFA titers of sows were ≦1:1000. Additionally, theserologic status of sows were from a known PRRS-negative herd.Twenty-eight (28) sows were tested for PCV2 serological status. Fourteen(14) sows had a PCV2 titer of ≦1000 and were transferred to the firststudy site. One hundred ten (110) piglets were delivered by cesareansection surgeries and were available for this study on Day −4. On Day−3, 108 CDCD pigs at the first study site were weighed, identified withear tags, blocked by weight and randomly assigned to 1 of 9 groups, asset forth above in table 4. If any test animal meeting the inclusioncriteria was enrolled in the study and was later excluded for anyreason, the Investigator and Monitor consulted in order to determine theuse of data collected from the animal in the final analysis. The date ofwhich enrolled piglets were excluded and the reason for exclusion wasdocumented. Initially, no sows were excluded. A total of 108 of anavailable 110 pigs were randomly assigned to one of 9 groups on Day −3.The two smallest pigs (No. 17 and 19) were not assigned to a group andwere available as extras, if needed. During the course of the study,several animals were removed. Pig 82 (Group 9) on Day −1, Pig No. 56(Group 6) on Day 3, Pig No. 53 (Group 9) on Day 4, Pig No. 28 (Group 8)on Day 8, Pig No. 69 (Group 8) on Day 7, and Pig No. 93 (Group 4) on Day9, were each found dead prior to challenge. These six pigs were notincluded in the final study results. Pig no 17 (one of the extra pigs)was assigned to Group 9. The remaining extra pig, No. 19, was excludedfrom the study.

The formulations given to each of the groups were as follows: Group 1was designed to administer 1 ml of viral ORF2 (vORF2) containing 16 μgORF2/ml. This was done by mixing 10.24 ml of viral ORF2 (256 μg/25μg/ml=10.24 ml vORF2) with 3.2 ml of 0.5% Carbopol and 2.56 ml ofphosphate buffered saline at a pH of 7.4. This produced 16 ml offormulation for group 1. Group 2 was designed to administer 1 ml ofvORF2 containing 8 μg vORF2/ml. This was done by mixing 5.12 ml of vORF2(128 μg/25 μg/ml=5.12 ml vORF2) with 3.2 ml of 0.5% Carbopol and 7.68 mlof phosphate buffered saline at a pH of 7.4. This produced 16 ml offormulation for group 2. Group 3 was designed to administer 1 ml ofvORF2 containing 4 μg vORF2/ml. This was done by mixing 2.56 ml of vORF2(64 μg/25 μg/ml=2.56 ml vORF2) with 3.2 ml of 0.5% Carbopol and 10.24 mlof phosphate buffered saline at a pH of 7.4. This produced 16 ml offormulation for group 3. Group 4 was designed to administer 1 ml ofrecombinant ORF2 (rORF2) containing 16 μg rORF2/ml. This was done bymixing 2.23 ml of rORF2 (512 μg/230 μg/ml=2.23 ml rORF2) with 6.4 ml of0.5% Carbopol and 23.37 ml of phosphate buffered saline at a pH of 7.4.This produced 32 ml of formulation for group 4. Group 5 was designed toadminister 1 ml of rORF2 containing 8 μg rORF2/ml. This was done bymixing 1.11 ml of rORF2 (256 μg/230 μg/ml=1.11 ml rORF2) with 6.4 ml of0.5% Carbopol and 24.49 ml of phosphate buffered saline at a pH of 7.4.This produced 32 ml of formulation for group 5. Group 6 was designed toadminister 1 ml of rORF2 containing 8 μg rORF2/ml. This was done bymixing 0.56 ml of rORF2 (128 μg/230 μg/ml=0.56 ml rORF2) with 6.4 ml of0.5% Carbopol and 25.04 ml of phosphate buffered saline at a pH of 7.4.This produced 32 ml of formulation for group 6. Group 7 was designed toadminister 2 ml of PCV2 whole killed cell vaccine (PCV2 KV) containingthe MAX PCV2 KV. This was done by mixing 56 ml of PCV2 KV with 14 ml of0.5% Carbopol. This produced 70 ml of formulation for group 7 Finallygroup 8 was designed to administer KLH at 0.5 μg/ml or 1.0 μg/ml per 2ml dose. This was done by mixing 40.71 ml KLH (7.0 μg protein/ml at 0.5μg/ml=570 ml (7.0 μg/ml)(x)=(0.5)(570 ml)), 244.29 ml phosphate bufferedsaline at a pH of 7.4, and 285 ml Freunds adjuvant. Table 5 describesthe time frames for the key activities of this Example.

TABLE 5 Study Activities Study Day Study Activity −4, 0 to 49 Generalobservations for overall health and clinical symptoms −3 Weighed;Randomized to groups; Collected blood samples from all pigs  0 Healthexamination; Administered IVP Nos. 1-7 to Groups 1-7, respectively 0-7Observed pigs for injection site reactions 14 Boostered Group 7 withPCV2 Vaccine No. 7; Blood samples from all pigs 14-21 Observed Group 7for injection site reactions 16-19 Treated all pigs with antibiotics(data missing) 19 Pigs transported from the first test site to a secondtest site 21 Treated Groups 1-9 with KLH/ICFA 24 Collected blood andnasal swab samples from all pigs; Weighed all pigs; Challenged Groups1-8 with PCV2 challenge material 25, 27, Collected nasal swab samplesfrom all pigs 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 27 Treated Groups1-9 with KLH/ICFA 31 Collected blood samples from all pigs 49 Collectedblood and nasal swab samples from all pigs; Weighed all pigs; Necropsyall pigs; Gross lesions noted with emphasis placed on icterus andgastric ulcers; Lungs evaluated for lesions; Fresh and formalin fixedtissue samples saved; In-life phase of the study completed

Following completion of the in-life phase of the study, formalin fixedtissues were examined by Immunohistochemistry (IHC) for detection ofPCV2 antigen by a pathologist, blood samples were evaluated for PCV2serology, nasal swab samples were evaluated for PCV2 shedding, andaverage daily weight gain (ADWG) was determined from Day 24 to Day 49.

Animals were housed at the first study site in individual cages in fiverooms from birth to approximately 11 days of age (approximately Day 0 ofthe study). Each room was identical in layout and consisted of stackedindividual stainless steel cages with heated and filtered air suppliedseparately to each isolation unit. Each room had separate heat andventilation, thereby preventing cross-contamination of air betweenrooms. Animals were housed in two different buildings at the secondstudy site. Group 9 (The Strict negative control group) was housedseparately in a converted finisher building and Groups 1-8 were housedin converted nursery building. Each group was housed in a separate pen(11-12 pigs per pen) and each pen provided approximately 3.0 square feetper pig. Each pen was on an elevated deck with plastic slatted floors. Apit below the pens served as a holding tank for excrement and waste.Each building had its own separate heating and ventilation systems, withlittle likelihood of cross-contamination of air between buildings.

At the first study site, piglets were fed a specially formulated milkration from birth to approximately 3 weeks of age. All piglets wereconsuming solid, special mixed ration by Day 19 (approximately 4½ weeksof age). At the second study site, all piglets were fed a customnon-medicated commercial mix ration appropriate for their age andweight, ad libitum. Water at both study sites was also available adlibitum.

All test pigs were treated with Vitamin E on Day −2, with ironinjections on Day −1 and with NAXCEL® (1.0 mL, IM, in alternating hams)on Days 16, 17, 18 and 19. In addition, Pig No. 52 (Group 9) was treatedwith an iron injection on Day 3, Pig 45 (Group 6) was treated with aniron injection on Day 11, Pig No. 69 (Group 8) was treated with NAXCEL®on Day 6, Pig No. 74 (Group 3) was treated with dexamethazone andpenicillin on Day 14, and Pig No. 51 (Group 1) was treated withdexamethazone and penicillin on Day 13 and with NAXCEL® on Day 14 forvarious health reasons.

While at both study sites, pigs were under veterinary care. Animalhealth examinations were conducted on Day 0 and were recorded on theHealth Examination Record Form. All animals were in good health andnutritional status before vaccination as determined by observation onDay 0. All test animals were observed to be in good health andnutritional status prior to challenge. Carcasses and tissues weredisposed of by rendering. Final disposition of study animals was recordson the Animal Disposition Record.

On Day 0, pigs assigned to Groups 1-6 received 1.0 mL of PCV2 Vaccines1-6, respectively, IM in the left neck region using a sterile 3.0 mLLuer-lock syringe and a sterile 20 g×½″ needle. Pigs assigned to Group 7received 2.0 mL of PCV2 Vaccine No. 7 IM in the left neck region using asterile 3.0 mL Luer-lock syringe and a sterile 20 g×½″ needle. On Day14, pigs assigned to Group 7 received 2.0 mL of PCV2 Vaccine No. 7 IM inthe right neck region using a sterile 3.0 mL Luer-lock syringe and asterile 20 g×½″ needle.

On Day 21 all test pigs received 2.0 mL of KLH/ICFA IM in the right hamregion using a sterile 3.0 mL Luer-lock syringe and a sterile 20 g×1″needle. On Day 27 all test pigs received 2.0 mL of KLH/ICFA in the leftham region using a sterile 3.0 mL Luer-lock syringe and a sterile 20g×1″ needle.

On Day 24, pigs assigned to Groups 1-8 received 1.0 mL of PCV2 ISUVDLchallenge material (5.11 log₁₀ TCID₅₀/mL) IM in the left neck regionusing a sterile 3.0 mL Luer-lock syringe and a sterile 20 g×1″ needle.An additional 1.0 mL of the same material was administered IN to eachpig (0.5 mL per nostril) using a sterile 3.0 mL Luer-lock syringe andnasal canula.

Test pigs were observed daily for overall health and adverse events onDay −4 and from Day 0 to Day 19. Observations were recorded on theClinical Observation Record. All test pigs were observed from Day 0 toDay 7, and Group 7 was further observed from Day 14 to 21, for injectionsite reactions. Average daily weight gain was determined by weighingeach pig on a calibrated scale on Days −3, 24 and 49, or on the day thata pig was found dead after challenge. Body weights were recorded on theBody Weight Form. Day −3 body weights were utilized to block pigs priorto randomization. Day 24 and Day 49 weight data was utilized todetermine the average daily weight gain (ADWG) for each pig during thesetime points. For pigs that died after challenge and before Day 49, theADWG was adjusted to represent the ADWG from Day 24 to the day of death.

In order to determine PCV2 serology, venous whole blood was collectedfrom each piglet from the orbital venous sinus on Days −3 and 14. Foreach piglet, blood was collected from the orbital venous sinus byinserting a sterile capillary tube into the medial canthus of one of theeyes and draining approximately 3.0 mL of whole blood into a 4.0 mLSerum Separator Tube (SST). On Days 24, 31, and 49, venous whole bloodfrom each pig was collected from the anterior vena cava using a sterile18 g×1½″ Vacutainer needle (Becton Dickinson and Company, FranklinLakes, N.J.), a Vacutainer needle holder and a 13 mL SST. Bloodcollections at each time point were recorded on the Sample CollectionRecord. Blood in each SST was allowed to clot, each SST was then spundown and the serum harvested. Harvested serum was transferred to asterile snap tube and stored at −70±10° C. until tested at a later date.Serum samples were tested for the presence of PCV2 antibodies byBIVI-R&D personnel.

Pigs were observed once daily from Day 20 to Day 49 for clinicalsymptoms and clinical observations were recorded on the ClinicalObservation Record.

To test for PCV2 nasal shedding, on Days 24, 25, and then every otherodd numbered study day up to and including Day 49, a sterile dacron swabwas inserted intra nasally into either the left or right nostril of eachpig (one swab per pig) as aseptically as possible, swished around for afew seconds and then removed. Each swab was then placed into a singlesterile snap-cap tube containing 1.0 mL of EMEM media with 2% IFBS, 500units/mL of Penicillin, 500 μg/mL of Streptomycin and 2.5 μg/mL ofFungizone. The swab was broken off in the tube, and the snap tube wassealed and appropriately labeled with animal number, study number, dateof collection, study day and “nasal swab.” Sealed snap tubes were storedat −40±10° C. until transported overnight on ice to BIVI-St. Joseph.Nasal swab collections were recorded on the Nasal Swab Sample CollectionForm. BIVI-R&D conducted quantitative virus isolation (VI) testing forPCV2 on nasal swab samples. The results were expressed in log₁₀ values.A value of 1.3 logs or less was considered negative and any valuegreater than 1.3 logs was considered positive.

Pigs that died (Nos. 28, 52, 56, 69, 82, and 93) at the first study sitewere necropsied to the level necessary to determine a diagnosis. Grosslesions were recorded and no tissues were retained from these pigs. Atthe second study site, pigs that died prior to Day 49 (Nos. 45, 23, 58,35), pigs found dead on Day 49 prior to euthanasia (Nos. 2, 43) and pigseuthanized on Day 49 were necropsied. Any gross lesions were noted andthe percentages of lung lobes with lesions were recorded on the NecropsyReport Form.

From each of the 103 pigs necropsied at the second study site, a tissuesample of tonsil, lung, heart, liver, mesenteric lymph node, kidney andinguinal lymph node was placed into a single container with buffered 10%formalin; while another tissue sample from the same aforementionedorgans was placed into a Whirl-pak (M-Tech Diagnostics Ltd., Thelwall,UK) and each Whirl-pak was placed on ice. Each container was properlylabeled. Sample collections were recorded on the Necropsy Report Form.Afterwards, formalin-fixed tissue samples and a Diagnostic Request Formwere submitted for IHC testing. IHC testing was conducted in accordancewith standard ISU laboratory procedures for receiving samples, sampleand slide preparation, and staining techniques. Fresh tissues inWhirl-paks were shipped with ice packs to the Study Monitor for storage(−70°±10° C.) and possible future use. Formalin-fixed tissues wereexamined by a pathologist for detection of PCV2 by IHC and scored usingthe following scoring system: 0=None; 1=Scant positive staining, fewsites; 2=Moderate positive staining, multiple sites; and 3=Abundantpositive staining, diffuse throughout the tissue. Due to the fact thatthe pathologist could not positively differentiate inguinal LN frommesenteric LN, results for these tissues were simply labeled as LymphNode and the score given the highest score for each of the two tissuesper animal.

Results

Results for this example are given below. It is noted that one pig fromGroup 9 died before Day 0, and 5 more pigs died post-vaccination (1 pigfrom Group 4; 1 pig from Group 6; 2 pigs from Group 8; and 1 pig fromGroup 9). Post-mortem examination indicated all six died due tounderlying infections that were not associated with vaccination or PMWS.Additionally, no adverse events or injection site reactions were notedwith any groups.

Average daily weight gain (ADWG) results are presented below in Table 6.Group 9, the strict negative control group, had the highest ADWG(1.06±0.17 lbs/day), followed by Group 5 (0.94±0.22 lbs/day), whichreceived one dose of 8 μg of rORF2. Group 3, which received one dose of4 μg of vORF2, had the lowest ADWG (0.49±0.21 lbs/day), followed byGroup 7 (0.50±0.15 lbs/day), which received 2 doses of killed vaccine.

TABLE 6 Summary of Group Average Daily Weight Gain (ADWG) ADWG - lbs/day(Day 24 to Day 49) or adjusted for Group Treatment N pigs dead beforeDay 29 1 vORF2 - 16 μg (1 dose) 12 0.87 ± 0.29 lbs/day 2 vORF2 - 8 μg (1dose) 12 0.70 ± 0.32 lbs/day 3 vORF2 - 4 μg (1 dose) 12 0.49 ± 0.21lbs/day 4 rORF2 - 16 μg (1 dose) 11 0.84 ± 0.30 lbs/day 5 rORF2 - 8 μg(1 dose) 12 0.94 ± 0.22 lbs/day 6 rORF2 - 4 μg (1 dose) 11 0.72 ± 0.25lbs/day 7 KV (2 doses) 12 0.50 ± 0.15 lbs/day 8 Challenge Controls 100.76 ± 0.19 lbs/day 9 Strict Negative Controls 11 1.06 ± 0.17 lbs/dayvORF2 = isolated viral ORF2; rORF2 = recombinant baculovirus expressedORF2; killed whole cell virus = PCV2 virus grown in suitable cellculture

PCV2 serology results are presented below in Table 7. All nine groupswere seronegative for PCV2 on Day −3. On Day 14, Groups receiving vORF2vaccines had the highest titers, which ranged from 187.5 to 529.2. Pigsreceiving killed viral vaccine had the next highest titers, followed bythe groups receiving rORF2 vaccines. Groups 8 and 9 remainedseronegative at this time. On Day 24 and Day 31, pigs receiving vORF2vaccines continued to demonstrate a strong serological response,followed closely by the group that received two doses of a killed viralvaccine. Pigs receiving rORF2 vaccines were slower to respondserologically and Groups 8 and 9 continued to remain seronegative. OnDay 49, pigs receiving vORF2 vaccine, 2 doses of the killed viralvaccine and the lowest dose of rORF2 demonstrated the strongestserological responses. Pigs receiving 16 μg and 8 μg of rORF2 vaccineshad slightly higher IFA titers than challenge controls. Group 9 on Day49 demonstrated a strong serological response.

TABLE 7 Summary of Group PCV2 IFA Titers Day Day Day Day Day GroupTreatment −3 14 24 31** 49*** 1 vORF2 - 16μg 50.0 529.2 4400.0 7866.711054.5 (1 dose) 2 vORF2 - 8 μg 50.0 500.0 3466.7 6800.0 10181.8 (1dose) 3 vORF2 - 4 μg 50.0 187.5 1133.3 5733.3 9333.3 (1 dose) 4 rORF2 -16μg 50.0 95.5 1550.0 3090.9 8000.0 (1 dose) 5 rORF2 - 8 μg 50.0 75.0887.5 2266.7 7416.7 (1 dose) 6 rORF2 - 4 μg 50.0 50.0 550.0 3118.210570.0 (1 dose) 7 KV (2 doses) 50.0 204.2 3087.5 4620.8 8680.0 8Challenge 50.0 55.0 50.0 50.0 5433.3 Controls 9 Strict Negative 50.059.1 59.1 54.5 6136.4 Controls vORF2 = isolated viral ORF2; rORF2 =recombinant baculovirus expressed ORF2; killed whole cell virus = PCV2virus grown in suitable cell culture *For calculation purposes, a ≦100IFA titer was designated as a titer of “50”; a ≧6400 IFA titer wasdesignated as a titer of “12,800”. **Day of Challenge ***Day of Necropsy

Average IFA Titer

The results from the post-challenge clinical observations are presentedbelow in Table 8. This summary of results includes observations forAbnormal Behavior, Abnormal Respiration, Cough and Diarrhea. Table 9includes the results from the Summary of Group Overall Incidence ofClinical Symptoms and Table 10 includes results from the Summary ofGroup Mortality Rates Post-challenge. The most common clinical symptomnoted in this study was abnormal behavior, which was scored as mild tosevere lethargy. Pigs receiving the 2 lower doses of vORF2, pigsreceiving 16 μg of rORF2 and pigs receiving 2 doses of KV vaccine hadincidence rates of ≧27.3%. Pigs receiving 8 μg of rORF2 and the strictnegative control group had no abnormal behavior. None of the pigs inthis study demonstrated any abnormal respiration. Coughing was notedfrequently in all groups (0 to 25%), as was diarrhea (0-20%). None ofthe clinical symptoms noted were pathognomic for PMWS.

The overall incidence of clinical symptoms varied between groups. Groupsreceiving any of the vORF2 vaccines, the group receiving 16 μg of rORF2,the group receiving 2 doses of KV vaccine and the challenge controlgroup had the highest incidence of overall clinical symptoms (≧36.4%).The strict negative control group, the group receiving 8 μg of rORF2 andthe group receiving 4 μg of rORF2 had overall incidence rates ofclinical symptoms of 0%, 8.3% and 9.1%, respectively.

Overall mortality rates between groups varied as well. The groupreceiving 2 doses of KV vaccine had the highest mortality rate (16.7%);while groups that received 4 μg of vORF2, 16 μg of rORF2, or 8 μg ofrORF2 and the strict negative control group all had 0% mortality rates.

TABLE 8 Summary of Group Observations for Abnormal Behavior, AbnormalRespiration, Cough, and Diarrhea Abnormal Abnormal Group Treatment NBehavior¹ Behavior² Cough³ Diarrhea⁴ 1 vORF2 - 16 μg (1 dose) 12 2/12(16.7%  0/12 (0%)  3/12 (25%)  2/12 (16/7%) 2 vORF2 - 8 μg (1 dose) 124/12 (33.3%) 0/12 (0%)  1/12 (8.3% 1/12 (8.3%) 3 vORF2 - 4 μg (1 dose)12 8/12 (66.7%) 0/12 (0%)   2/12 (16.7%) 1/12 (8.3%) 4 rORF2 - 16 μg (1dose) 11 3/11 (27.3%) 0/11 (0%) 0/11 (0%)  2/11 (18.2%) 5 rORF2 - 8 μg(1 dose) 12 0/12 (0%)   0/12 (0%)  1/12 (8.3%) 0/12 (0%)  6 rORF2 - 4 μg(1 dose) 11 1/11 (9.1%)  0/11 (0%) 0/11 (0%) 0/12 (0%)  7 KV (2 doses)12 7/12 (58.3)  0/12 (0%) 0/12 (0%) 1/12 (8.3%) 8 Challenge Controls 101/10 (10%)  0/10 (0%)  2/10 (20%) 2/10 (20%)  9 Strict Negative Controls11 0/11 (0%)   0/11 (0%) 0/11 (0%) 0/11 (0%)  vORF2 = isolated viralORF2; rORF2 = recombinant baculovirus expressed ORF2; killed whole cellvirus = PCV2 virus grown in suitable cell culture ¹Total number of pigsin each group that demonstrated any abnormal behavior for at least oneday ²Total number of pigs in each group that demonstrated any abnormalrespiration for at least one day ³Total number of pigs in each groupthat demonstrated a cough for at least one day ⁴Total number of pigs ineach group that demonstrated diarrhea for at least one day

TABLE 9 Summary of Group Overall Incidence of Clinical SymptomsIncidence of pigs with Incidence Group Treatment N Clinical Symptoms¹Rate 1 vORF2 - 16 μg (1 dose) 12 5 41.7% 2 vORF2 - 8 μg (1 dose) 12 541.7% 3 vORF2 - 4 μg (1 dose) 12 8 66.7% 4 rORF2 - 16 μg (1 dose) 11 436.4% 5 rORF2 - 8 μg (1 dose) 12 1  8.3% 6 rORF2 - 4 μg (1 dose) 11 1 9.1% 7 KV (2 doses) 12 7 58.3% 8 Challenge Controls 10 4   40% 9 StrictNegative 11 0   0% Controls vORF2 = isolated viral ORF2; rORF2 =recombinant baculovirus expressed ORF2; killed whole cell virus = PCV2virus grown in suitable cell culture ¹Total number of pigs in each groupthat demonstrated any clinical symptom for at least one day

TABLE 10 Summary of Group Mortality Rates Post - challenge Dead Post-Group Treatment N challenge Mortality Rate 1 vORF2 - 16 μg (1 dose) 12 18.3% 2 vORF2 - 8 μg (1 dose) 12 1 8.3% 3 vORF2 - 4 μg (1 dose) 12 0   0%4 rORF2 - 16 μg (1 dose) 11 0   0% 5 rORF2 - 8 μg (1 dose) 12 0   0% 6rORF2 - 4 μg (1 dose) 11 1 9.1% 7 KV (2 doses) 12 2 16.7%  8 ChallengeControls 10 1  10% 9 Strict Negative Controls 11 0   0% vORF2 = isolatedviral ORF2; rORF2 = recombinant baculovirus expressed ORF2; killed wholecell virus = PCV2 virus grown in suitable cell culture

PCV2 nasal shedding results are presented below in Table 11. Followingchallenge on Day 24, 1 pig in Group 7 began shedding PCV2 on Day 27.None of the other groups experienced shedding until Day 33. The bulk ofnasal shedding was noted from Day 35 to Day 45. Groups receiving any ofthe three vORF2 vaccines and groups receiving either 4 or 8 μg of rORF2had the lowest incidence of nasal shedding of PCV2 (≦9.1%). Thechallenge control group (Group 8) had the highest shedding rate (80%),followed by the strict negative control group (Group 9), which had anincidence rate of 63.6%.

TABLE 11 Summary of Group Incidence of Nasal Shedding of PCV2 No. Ofpigs that shed Incidence Group Treatment N for at least one day Rate 1vORF2 - 16 μg (1 dose) 12 1 8.3% 2 vORF2 - 8 μg (1 dose) 12 1 8.3% 3vORF2 - 4 μg (1 dose) 12 1 8.3% 4 rORF2 - 16 μg (1 dose) 11 2 18.2%  5rORF2 - 8 μg (1 dose) 12 1 8.3% 6 rORF2 - 4 μg (1 dose) 11 1 9.1% 7 KV(2 doses) 12 5 41.7%  8 Challenge Controls 10 8  80% 9 Strict Negative11 7 63.6%  Controls vORF2 = isolated viral ORF2; rORF2 = recombinantbaculovirus expressed ORF2; killed whole cell virus = PCV2 virus grownin suitable cell culture

The Summary of Group Incidence of Icterus, Group Incidence of GastricUlcers, Group Mean Lung Lesion Scores, and Group Incidence of LungLesions are shown below in Table 12. Six pigs died at the first testsite during the post-vaccination phase of the study (Group 4, N=1; Group6, N=1; Group 8, N=2; Group 9, N=2). Four out of six pigs had fibrinouslesions in one or more body cavities, one pig (Group 6) had lesionsconsistent with clostridial disease, and one pig (Group 9) had no grosslesions. None of the pigs that died during the post-vaccination phasedof the study had lesions consistent with PMWS.

Pigs that died post-challenge and pigs euthanized on Day 49 werenecropsied. At necropsy, icterus and gastric ulcers were not present inany group. With regard to mean % lung lesions, Group 9 had lowest mean %lung lesions (0%), followed by Group 1 with 0.40±0.50% and Group 5 with0.68±1.15%. Groups 2, 3, 7 and 8 had the highest mean % lung lesions(≧7.27%). Each of these four groups contained one pig with % lunglesions ≧71.5%, which skewed the results higher for these four groups.With the exception of Group 9 with 0% lung lesions noted, the remaining8 groups had ≦36% lung lesions. Almost all lung lesions noted weredescribed as red/purple and consolidated.

TABLE 12 Summary of Group Incidence of Icterus, Group Incidence ofGastric Ulcers, Group Mean % Lung Lesion Scores, and Group Incidence ofLung Lesions Noted Incidence of Gastric Mean % Lung Lung Lesions GroupTreatment Icterus Ulcers Lesions Noted 1 vORF2 - 16 μg (1 0/12 (0%) 0/120.40 ± 0.50% 10/12  dose) (0%) (83%) 2 vORF2 - 8 μg (1 dose) 0/12 (0%)0/12 7.41 ± 20.2% 10/12  (0%) (83%) 3 vORF2 - 4 μg (1 dose) 0/12 (0%)0/12 9.20 ± 20.9% 10/12  (0%) (83%) 4 rORF2 - 16 μg (1 0/11 (0%) 0/11 1.5 ± 4.74% 4/11 dose) (0%) (36%) 5 rORF2 - 8 μg (1 dose) 0/12 (0%)0/12 0.68 ± 1.15% 9/12 (0%) (75%) 6 rORF2 - 4 μg (1 dose) 0/11 (0%) 0/112.95 ± 5.12% 7/11 (0%) (64%) 7 KV (2 doses) 0/12 (0%) 0/12 7.27 ± 22.9%9/12 (0%) (75%) 8 Challenge Controls 0/10 (0%) 0/10 9.88 ± 29.2% 8/10(0%) (80%) 9 Strict Negative 0/11 (0%) 0/11 0/11 0/11 Controls (0%) (0%) (0%) vORF2 = isolated viral ORF2; rORF2 = recombinant baculovirusexpressed ORF2; KV or killed whole cell virus = PCV2 virus grown insuitable cell culture

The Summary of Group IHC Positive Incidence Results are shown in Table13. Group 1 (vORF2-16 μg) and Group 5 (rORF2-8 μg) had the lowest rateof IHC positive results (16.7%). Group 8 (Challenge Controls) and Group9 (Strict Negative Controls) had the highest rate of IHC positiveresults, 90% and 90.9%, respectively.

TABLE 13 Summary of Group IHC Positive Incidence Rate No. Of pigs thathad at least one tissue Incidence Group Treatment N positive for PCV2Rate 1 vORF2 - 16 μg (1 dose) 12 2 16.7% 2 vORF2 - 8 μg (1 dose) 12 325.0% 3 vORF2 - 4 μg (1 dose) 12 8 66.7% 4 rORF2 - 16 μg (1 dose) 11 436.3% 5 rORF2 - 8 μg (1 dose) 12 2 16.7% 6 rORF2 - 4 μg (1 dose) 11 436.4% 7 KV (2 doses) 12 5 41.7% 8 Challenge Controls 10 9 90.0% 9 StrictNegative Controls 11 10 90.9% vORF2 = isolated viral ORF2; rORF2 =recombinant baculovirus expressed ORF2; KV or killed whole cell virus =PCV2 virus grown in suitable cell culture

Post-challenge, Group 5, which received one dose of 8 μg of rORF2antigen, outperformed the other 6 vaccine groups. Group 5 had thehighest ADWG (0.94±0.22 lbs/day), the lowest incidence of abnormalbehavior (0%), the second lowest incidence of cough (8.3%), the lowestincidence of overall clinical symptoms (8.3%), the lowest mortality rate(0%), the lowest rate of nasal shedding of PCV2 (8.3%), the secondlowest rate for mean % lung lesions (0.68±1.15%) and the lowestincidence rate for positive tissues (16.7%). Groups receiving variouslevels of rORF2 antigen overall outperformed groups receiving variouslevels of vORF2 and the group receiving 2 doses of killed whole cellPCV2 vaccine performed the worst. Tables 14 and 15 contain summaries ofgroup post-challenge data.

TABLE 14 Summary of Group Post-Challenge Data - Part 1 ADWG AbnormalOverall Incidence Group N Treatment (lbs/day) Behavior Cough of ClinicalSymptoms 1 12 vORF2 - 16 μg 0.87 ± 0.29 2/12 (16.7%)  3/12 (25%) 41.7%(1 dose) 2 12 vORF2 - 8 μg 0.70 ± 0.32 4/12 (33.3%   1/12 (8.3% 41.7% (1dose) 3 12 vORF2 - 4 μg 0.49 ± 0.21 8/12 (66.7%)  2/12 (16.7% 66.7% (1dose) 4 11 rORF2 - 16 μg 0.84 ± 0.30 3/11 (27.3%) 0/11 (0%) 36.4% (1dose) 5 12 rORF2 - 8 μg 0.94 ± 0.22 0/12 (0%)    1/12 (8.3%  8.3% (1dose) 6 11 rORF2 - 4 μg 0.72 ± 0.25 1/11 (9.1%  0/11 (0%)  9.1% (1 dose)7 12 KV 0.50 ± 0.15 7/12 (58.3)  0/12 (0%) 58.3% (2 doses) 8 10Challenge 0.76 ± 0.19 1/10 (10%)   2/10 (20%  40% Controls 9 11 StrictNegative 1.06 + 0.17 0/11 (0%)   0/11 (0%)   0% Controls vORF2 =isolated viral ORF2; rORF2 = recombinant baculovirus expressed ORF2; KVor killed whole cell virus = PCV2 virus grown in suitable cell culture

TABLE 15 Summary of Group Post-Challenge Data - Part 2 Mean % IncidenceRate of at Mortality Nasal Lung least one tissue IHC Group N TreatmentRate Shedding Lesions positive for PCV2 1 12 vORF2 - 16 μg 8.3%  8.3%0.40 ± 0.50% 16.7% (1 dose) 2 12 vORF2 - 8 μg 8.3%  8.3% 7.41 ± 20.2%25.0% (1 dose) 3 12 vORF2 - 4 μg 0% 8.3% 9.20 ± 20.9% 66.7% (1 dose) 411 rORF2 - 16 μg 0% 18.2%  1.50 ± 4.74% 36.3% (1 dose) 5 12 rORF2 - 8 μg0% 8.3% 0.68 ± 1.15% 16.7% (1 dose) 6 11 rORF2 - 4 μg 9.1%  9.1% 2.95 ±5.12% 36.4% (1 dose) 7 12 KV 16.7%   41.7%  7.27 ± 22.9% 41.7% (2 doses)8 10 Challenge 10%   80% 9.88 ± 29.2% 90.0% Controls 9 11 StrictNegative 0% 63.6%  0/11 (0%) 90.9% Controls vORF2 = isolated viral ORF2;rORF2 = recombinant baculovirus expressed ORF2; KV or killed whole cellvirus = PCV2 virus grown in suitable cell culture

Results of this study indicate that all further vaccine efforts shouldfocus on a rORF2 vaccine. Overall, nasal shedding of PCV2 was detectedpost-challenge and vaccination with a PCV2 vaccine resulted in areduction of shedding. Immunohistochemistry of selected lymphoid tissuesalso served as a good parameter for vaccine efficacy, whereas largedifferences in ADWG, clinical symptoms, and gross lesions were notdetected between groups. This study was complicated by the fact thatextraneous PCV2 was introduced at some point during the study, asevidenced by nasal shedding of PCV2, PCV2 seroconversion and positiveIHC tissues in Group 9, the strict negative control group.

Discussion

Seven PCV2 vaccines were evaluated in this study, which included threedifferent dose levels of vORF2 antigen administered once on Day 0, threedifferent dose levels of rORF2 antigen administered once on Day 0 andone dose level of killed whole cell PCV2 vaccine administered on Day 0and Day 14. Overall, Group 5, which received 1 dose of vaccinecontaining 8 μg of rORF2 antigen, had the best results. Group 5 had thehighest ADWG, the lowest incidence of abnormal behavior, the lowestincidence of abnormal respiration, the second lowest incidence of cough,the lowest incidence of overall clinical symptoms, the lowest mortalityrate, the lowest rate of nasal shedding of PCV2, the second lowest ratefor mean % lung lesions and the lowest incidence rate for positive IHCtissues.

Interestingly, Group 4, which received a higher dose of rORF2 antigenthan Group 5, did not perform as well or better than Group 5. Group 4had a slightly lower ADWG, a higher incidence of abnormal behavior, ahigher incidence of overall clinical symptoms, a higher rate of nasalshedding of PCV2, a higher mean % lung lesions, and a higher rate forpositive IHC tissues than Group 5. Statistical analysis, which may haveindicated that the differences between these two groups were notstatistically significant, was not conducted on these data, but therewas an observed trend that Group 4 did not perform as well as Group 5.

Post-vaccination, 6 pigs died at the first study site. Four of the sixpigs were from Group 8 or Group 9, which received no vaccine. None ofthe six pigs demonstrated lesions consistent with PMWS, no adverseevents were reported and overall, all seven vaccines appeared to be safewhen administered to pigs approximately 11 days of age. During thepost-vaccination phase of the study, pigs receiving either of three doselevels of vORF2 vaccine or killed whole cell vaccine had the highestIFAT levels, while Group 5 had the lowest IFAT levels just prior tochallenge, of the vaccine groups.

Although not formally proven, the predominant route of transmission ofPCV2 to young swine shortly after weaning is believed to be by oronasaldirect contact and an efficacious vaccine that reduces nasal shedding ofPCV2 in a production setting would help control the spread of infection.Groups receiving one of three vORF2 antigen levels and the groupreceiving 8 μg of rORF2 had the lowest incidence rate of nasal sheddingof PCV2 (8.3%). Expectedly, the challenge control group had the highestincidence rate of nasal shedding (80%).

Gross lesions in pigs with PMWS secondary to PCV2 infection typicallyconsist of generalized lymphadenopathy in combination with one or amultiple of the following: (1) interstitial pneumonia with interlobularedema, (2) cutaneous pallor or icterus, (3) mottled atrophic livers, (4)gastric ulcers and (5) nephritis. At necropsy, icterus, hepatitis,nephritis, and gastric ulcers were not noted in any groups andlymphadenopathy was not specifically examined for. The mean % lunglesion scores varied between groups. The group receiving 16 μg of vORF2antigen had the lowest mean % lung lesion score (0.40±0.50%), followedby the group that received 8 μg of rORF2 (0.68±1.15%). As expected, thechallenge control group had the highest mean % lung lesion score(9.88±29.2%). In all four groups, the mean % lung lesion scores wereelevated due to one pig in each of these groups that had very high lunglesion scores. Most of the lung lesions were described as red/purple andconsolidated. Typically, lung lesions associated with PMWS are describedas tan and non-collapsible with interlobular edema. The lung lesionsnoted in this study were either not associated with PCV2 infection or asecond pulmonary infectious agent may have been present. Within thecontext of this study, the % lung lesion scores probably do not reflecta true measure of the amount of lung infection due to PCV2.

Other researchers have demonstrated a direct correlation between thepresence of PCV2 antigen by IHC and histopathology. Histopathology onselect tissues was not conducted with this study. Group 1 (16 μg ofvORF2) and Group 5 (8 μg of rORF2) had the lowest incidence rate of pigspositive for PCV2 antigen (8.3%), while Group 9 (the strict negativecontrol group—90.9%) and Group 8 (the challenge control group—90.0%) hadthe highest incidence rates for pigs positive for PCV2 antigen. Due tothe non-subjective nature of this test, IHC results are probably one ofthe best parameters to judge vaccine efficacy on.

Thus, in one aspect of the present invention, the Minimum PortectiveDosage (MPD) of a 1 ml/1 dose recombinant product with extracted PCV2ORF2 (rORF2) antigen in the CDCD pig model in the face of a PCV2challenge was determined Of the three groups that received varyinglevels of rORF2 antigen, Group 5 (8 μg of rORF2 antigen) clearly had thehighest level of protection. Group 5 either had the best results or wastied for the most favorable results with regard to all of the parametersexamined. When Group 5 was compared with the other six vaccine groupspost-challenge, Group 5 had the highest ADWG (0.94±0.22 lbs/day), thelowest incidence of abnormal behavior (0%), the second lowest incidenceof cough (8.3%), the lowest incidence of overall clinical symptoms(8.3%), the lowest mortality rate (0%), the lowest rate of nasalshedding of PCV2 (8.3%), the second lowest rate for mean % lung lesions(0.68±1.15%) and the lowest incidence rate for positive IHC tissues(16.7%).

In another aspect of the present invention, the MPD of a 1 ml/1 doseconventional product that is partially purified PCV2 ORF2 (vORF2)antigen in the CDCD pig model in the face of a PCV2 challenge wasdetermined Of the three groups that received varying levels of vORF2antigen, Group 1 (16 μg of vORF2) had the highest level of protection.Group 1 outperformed Groups 2 and 3 with respect to ADWG, mean % lunglesions, and IHC. Groups 1 and 2 (8 μg of vORF2 antigen) performedequally with respect to overall incidence of clinical symptoms, Group 3(4 μg of vORF2 antigen) had the lowest mortality rate, and all threegroups performed equally with respect to nasal shedding. Overall, vORFvaccines did not perform as well as rORF vaccines.

In yet another aspect of the present invention, the efficacy of amaximum dose of a 2 ml/2 dose Conventional Killed PCV2 vaccine in theCDCD pig model in the face of a PCV2 challenge was determined Of theseven vaccines evaluated in this study, the killed whole cell PCV2vaccine performed the worst. Piglets receiving two doses of killed wholecell PCV2 vaccine had the lowest ADWG, the second highest rate ofabnormal behavior (58.3%), the second highest overall incidence ofclinical symptoms (58.3%), the highest mortality rate (16.7%), thesecond highest incidence of nasal shedding (41.7%), highest mean % lunglesions (9.88±29.2%), a high incidence of lung lesions noted (75%) and amoderate IHC incidence rate in tissues (41.7%). However, it was stilleffective at invoking an immune response.

In still another aspect of the present invention, nasal shedding of PCV2was assessed as an efficacy parameter and the previous PCV2 efficacyparameters from previous studies were reconfirmed. Results from thisstudy indicate that nasal shedding of PCV2 occurs following intra nasalchallenge and that PCV2 vaccines reduce nasal shedding of PCV2post-challenge. Furthermore, results from this study and reports in theliterature indicate that IHC should continue to be evaluated in futurePCV2 vaccine trials as well.

Some additional conclusions arising from this study are thatlymphadenopathy is one of the hallmarks of PMWS. Another one of thehallmarks of PMWS is lymphoid depletion and multinucleated/gianthistiocytes. Additionally, no adverse events or injection site reactionswere noted for any of the 7 PCV2 vaccines and all 7 PCV2 vaccinesappeared to be safe when administered to young pigs.

Example 5

This example tests the efficacy of eight PCV2 candidate vaccines andreconfirms PCV2 challenge parameters from earlier challenge studiesfollowing exposure to a virulent strain of PCV2. One hundred and fifty(150) cesarean derived colostrum deprived (CDCD) piglets, 6-16 days ofage, were blocked by weight and randomly divided into 10 groups of equalsize. Table 16 sets forth the General Study Design for this Example.

TABLE 16 General Study Design KLH/ICFA Challenge PRRSV Necropsy No. OfDay of on Day 22 with Virulent MLV on on Day Group Pigs TreatmentTreatment and Day 28 PCV2 on Day 25 Day 46 50 1 15 PVC2 Vaccine 1 16 μg0 & 14 + + + + rORF2 - IMS 1314 2 15 PVC2 Vaccine 2 16 μg 0 & 14 + + + +vORF2 - Carbopol 3 15 PCV2 Vaccine 3 16 μg 0 & 14 + + + + rORF2 -Carbopol 4 15 PCV2 Vaccine 2 16 μg 0 + + + + vORF2 - Carbopol 5 15 PVC2Vaccine 3 4 μg 0 & 14 + + + + rORF2 - Carbopol 6 15 PVC2 Vaccine 3 1 μg0 & 14 + + + + rORF2 - Carbopol 7 15 PVC2 Vaccine 3 0.25 μg 0 &14 + + + + rORF2 - Carbopol 8 15 PVC2 Vaccine 4 > 8.0 log 0 & 14 + + + +KV - Carbopol 9 15 Challenge Controls N/A + + + + 10 15 None - StrictN/A + − + + Negative Control Group vORF2 = isolated viral ORF2; rORF2 =recombinant baculovirus expressed ORF2; KV or killed whole cell virus =PCV2 virus grown in suitable cell culture

The vaccine formulation given to each group were as follows. PCV2Vaccine No. 1, administered at 1×2 ml dose to Group 1, was a high dose(16 ug/2 ml dose) of inactivated recombinant ORF2 antigen adjuvantedwith IMS 1314 (16 ug rORF2—IMS 1314). PCV2 Vaccine No. 2, administeredat 1×2 ml dose to Group 2, was a high dose (16 ug/2 ml dose) of apartially purified VIDO R-1 generated PCV2 ORF2 antigen adjuvanted withCarbopol (16 ug vORF2—Carbopol). PCV2 Vaccine No. 3, administered at 1×2ml dose to Group 3, was a high dose (16 ug/2 ml dose) of inactivatedrecombinant ORF2 antigen adjuvanted with Carbopol (16 ugrORF2—Carbopol). PCV2 Vaccine No. 4, administered at 1×1 ml dose toGroup 4, was a high dose (16 ug/1 ml dose) of a partially purified VIDOR-1 generated PCV2 ORF2 antigen adjuvanted with Carbopol (16 ugvORF2—Carbopol). Vaccine No. 5, administered at 1×2 ml dose to Group 5,was a 4 ug/2 ml dose of an inactivated recombinant ORF2 antigenadjuvanted with Carbopol (4 ug rORF2—Carbopol). PCV2 Vaccine No. 6,administered at 1×2 ml dose to Group 6, was a 1 ug/2 ml dose of aninactivated recombinant ORF2 antigen adjuvanted with Carbopol (1 ugrORF2—Carbopol). PCV2 Vaccine No. 7, administered at 1×2 ml dose toGroup 7, was a low dose (0.25 ug/2 ml dose) of inactivated recombinantORF2 antigen adjuvanted with Carbopol (0.25 ug rORF2—Carbopol). PCV2Vaccine No. 8, administered at 1×2 ml dose to Group 8, was a high dose(pre-inactivation titer >8.0 log/2 ml dose) Inactivated ConventionalKilled VIDO R-1 generated PCV2 Struve antigen adjuvanted with Carbopol(>8.0 log KV—Carbopol). On Day 0, Groups 1-8 were treated with theirassigned vaccines. Groups 1-3 and 5-8 received boosters of theirrespective vaccines again on Day 14. The effectiveness of a single doseof 16 μg of vORF2—Carbopol was tested on Group 4 which did not receive abooster on Day 14. Piglets were observed for adverse events andinjection site reactions following both vaccinations. On Day 21 thepiglets were moved to a second study site where Groups 1-9 were grouphoused in one building and Group 10 was housed in a separate building.All pigs received keyhole limpet hemocyanin emulsified with incompleteFreund's adjuvant (KLH/ICFA) on Days 22 and 28. On Day 25, Groups 1-9were challenged with approximately 4 logs of virulent PCV2 virus. By Day46, very few deaths had occurred in the challenge control group. In anattempt to immunostimulate the pigs and increase the virulence of thePCV2 challenge material, all Groups were treated with INGELVAC® PRRSVMLV (Porcine Reproductive and Respiratory Vaccine, Modified Live Virus)on Day 46.

Pre- and post-challenge blood samples were collected for PCV2 serology.Post-challenge, body weight data for determination of average dailyweight gain (ADWG) and observations of clinical signs were collected. OnDay 50, all surviving pigs were necropsied, gross lesions were recorded,lungs were scored for pathology, and selected tissues were preserved informalin for examination by Immunohistochemistry (IHC) for detection ofPCV2 antigen at a later date.

Materials and Methods

This was a partially-blind vaccination-challenge feasibility studyconducted in CDCD pigs, 6 to 16 days of age on Day 0. To be included inthe study, PCV2 IFA titers of sows were ≦1:1000. Additionally, theserologic status of sows were from a known PRRS-negative herd. Sixteen(16) sows were tested for PCV2 serological status and all sixteen (16)had a PCV2 titer of ≦1000 and were transferred to the first study site.One hundred fifty (150) piglets were delivered by cesarean sectionsurgeries and were available for this study on Day −3. On Day −3, 150CDCD pigs at the first study site were weighed, identified with eartags, blocked by weight and randomly assigned to 1 of 10 groups, as setforth above in table 16. Blood samples were collected from all pigs. Ifany test animal meeting the inclusion criteria was enrolled in the studyand was later excluded for any reason, the Investigator and Monitorconsulted in order to determine the use of data collected from theanimal in the final analysis. The date of which enrolled piglets wereexcluded and the reason for exclusion was documented. No sows meetingthe inclusion criteria, selected for the study and transported to thefirst study site were excluded. No piglets were excluded from the study,and no test animals were removed from the study prior to termination.Table 17 describes the time frames for the key activities of thisExample.

TABLE 17 Study Activities Study Day Actual Dates Study Activity −3 Apr.04, 2003 Weighed pigs; health exam; randomized to groups; collectedblood samples −3, Apr. 04, 2003 Observed for overall health and foradverse events post-  0-21 Apr. 07, 2003 to vaccination May 27, 2003  0Apr. 07, 2003 Administered respective IVPs to Groups 1-8 0-7 Apr. 07,2003 to Observed pigs for injection site reactions Apr. 14, 2003 14 Apr.21, 2003 Boostered Groups 1-3, 5-8 with respective IVPs; blood sampledall pigs 14-21 Apr. 21, 2003 to Observed pigs for injection reactionsApr. 28, 2003 19-21 Apr. 26, 2003 to Treated all pigs with antibioticsApr. 28, 2003 21 Apr. 28, 2003 Pigs transported from Struve Labs, Inc.to Veterinary Resources, Inc. (VRI) 22-50 Apr. 28, 2003 to Observed pigsfor clinical signs post-challenge May 27, 2003 22 Apr. 29, 2003 TreatedGroups 1-10 with KLH/ICFA 25 May 02, 2003 Collected blood samples fromall pigs; weighed all pigs; challenged Groups 1-9 with PCV2 challengematerial 28 May 05, 2003 Treated Groups 1-10 with KLH/ICFA 32 May 09,2003 Collected blood samples from all pigs 46 May 23, 2003 AdministeredINGELVAC ® PRRS MLV to all groups 50 May 27, 2003 Collected bloodsamples, weighed and necropsied all pigs; gross lesions were recorded;lungs were evaluated for lesions; fresh and formalin fixed tissuesamples were saved; In-life phase of the study was completed

Following completion of the in-life phase of the study, formalin fixedtissues were examined by Immunohistochemistry (IHC) for detection ofPCV2 antigen by a pathologist, blood samples were evaluated for PCV2serology, and average daily weight gain (ADWG) was determined from Day25 to Day 50.

Animals were housed at the first study site in individual cages in sevenrooms from birth to approximately 11 days of age (approximately Day 0 ofthe study). Each room was identical in layout and consisted of stackedindividual stainless steel cages with heated and filtered air suppliedseparately to each isolation unit. Each room had separate heat andventilation, thereby preventing cross-contamination of air betweenrooms. Animals were housed in two different buildings at the secondstudy site. Group 10 (The Strict negative control group) was housedseparately in a converted nursery building and Groups 1-9 were housed ina converted farrowing building. Each group was housed in a separate pen(14-15 pigs per pen) and each pen provided approximately 2.3 square feetper pig. Groups 2, 4 and 8 were penned in three adjacent pens on oneside of the alleyway and Groups 1, 3, 5, 6, 7, and 9 were penned in sixadjacent pens on the other side of the alleyway. The Group separationwas due to concern by the Study Monitor that vaccines administered toGroups 2, 4, and 8 had not been fully inactivated. Each pen was on anelevated deck with plastic slatted floors. A pit below the pens servedas a holding tank for excrement and waste. Each building had its ownseparate heating and ventilation systems, with little likelihood ofcross-contamination of air between buildings.

At the first study site, piglets were fed a specially formulated milkration from birth to approximately 3 weeks of age. All piglets wereconsuming solid, special mixed ration by Day 21 (approximately 4½ weeksof age). At the second study site, all piglets were fed a customnon-medicated commercial mix ration appropriate for their age andweight, ad libitum. Water at both study sites was also available adlibitum.

All test pigs were treated with 1.0 mL of NAXCEL®, IM, in alternatinghams on Days 19, 20, and 21. In addition, Pig No. 11 (Group 1) wastreated with 0.5 mL of NAXCEL® IM on Day 10, Pig No. 13 (Group 10) wastreated with 1 mL of Penicillin and 1 mL of PREDEF® 2× on Day 10, PigNo. 4 (Group 9) was treated with 1.0 mL of NAXCEL® IM on Day 11, andPigs 1 (Group 1), 4 and 11 were each treated with 1.0 mL of NAXCEL® onDay 14 for various health reasons.

While at both study sites, pigs were under veterinary care. Animalhealth examinations were conducted on Day −3 and were recorded on theHealth Examination Record Form. All animals were in good health andnutritional status before vaccination as determined by observation onDay 0. All test animals were observed to be in good health andnutritional status prior to challenge. Carcasses and tissues weredisposed of by rendering. Final disposition of study animals wasrecorded on the Animal Disposition Record.

On Days 0 and 14, pigs assigned to Groups 1-3 and 5-8 received 2.0 mL ofassigned PCV2 Vaccines 1-4, respectively, IM in the right and left neckregion, respectively, using a sterile 3.0 mL Luer-lock syringe and asterile 20 g×½″ needle. Pigs assigned to Group 4 received 1.0 mL of PCV2Vaccine No. 2, IM in the right neck region using a sterile 3.0 mLLuer-lock syringe and a sterile 20 g×½″ needle on Day 0 only.

On Day 22 all test pigs received 2.0 mL of KLH/ICFA IM in the left neckregion using a sterile 3.0 mL Luer-lock syringe and a sterile 20 g×1″needle. On Day 28 all test pigs received 2.0 mL of KLH/ICFA in the rightham region using a sterile 3.0 mL Luer-lock syringe and a sterile 20g×1″ needle.

On Day 25, pigs assigned to Groups 1-9 received 1.0 mL of PCV2 ISUVDLchallenge material (3.98 log₁₀ TCID₅₀/mL) IM in the right neck regionusing a sterile 3.0 mL Luer-lock syringe and a sterile 20 g×1″ needle.An additional 1.0 mL of the same material was administered IN to eachpig (0.5 mL per nostril) using a sterile 3.0 mL Luer-lock syringe andnasal canula.

On Day 46, all test pigs received 2.0 mL INGELVAC® PRRS MLV, IM, in theright neck region using a sterile 3.0 mL Luer0lock syringe and a sterile20 g×1″ needle. The PRRSV MLV was administered in an attempt to increasevirulence of the PCV2 challenge material.

Test pigs were observed daily for overall health and adverse events onDay −3 and from Day 0 to Day 21. Each of the pigs were scored for normalor abnormal behavior, respiration or cough. Observations were recordedon the Clinical Observation Record. All test pigs were observed from Day0 to Day 7, and Group 7 was further observed from Day 14 to 21, forinjection site reactions. Average daily weight gain was determined byweighing each pig on a calibrated scale on Days −3, 25 and 50, or on theday that a pig was found dead after challenge. Body weights wererecorded on the Body Weight Form. Day −3 body weights were utilized toblock pigs prior to randomization. Day 25 and Day 50 weight data wasutilized to determine the average daily weight gain (ADWG) for each pigduring these time points. For pigs that died after challenge and beforeDay 50, the ADWG was adjusted to represent the ADWG from Day 25 to theday of death.

In order to determine PCV2 serology, venous whole blood was collectedfrom each piglet from the orbital venous sinus on Days −3 and 14. Foreach piglet, blood was collected from the orbital venous sinus byinserting a sterile capillary tube into the medial canthus of one of theeyes and draining approximately 3.0 mL of whole blood into a 4.0 mLSerum Separator Tube (SST). On Days 25, 32, and 50, venous whole bloodfrom each pig was collected from the anterior vena cava using a sterile20 g×1½″ Vacutainer® needle (Becton Dickinson and Company, FranklinLakes, N.J.), a Vaccutainer® needle holder and a 13 mL SST. Bloodcollections at each time point were recorded on the Sample CollectionRecord. Blood in each SST was allowed to clot, each SST was then spundown and the serum harvested. Harvested serum was transferred to asterile snap tube and stored at −70±10° C. until tested at a later date.Serum samples were tested for the presence of PCV2 antibodies byBIVI-R&D personnel.

Pigs were observed once daily from Day 22 to Day 50 for clinicalsymptoms and scored for normal or abnormal behavior, respiration orcough. Clinical observations were recorded on the Clinical ObservationRecord.

Pigs Nos. 46 (Group 1) and 98 (Groups 9) died at the first study site.Both of these deaths were categorized as bleeding deaths and necropsieswere not conducted on these two pigs. At the second study site, pigsthat died after challenge and prior to Day 50, and pigs euthanized onDay 50, were necropsied. Any gross lesions were noted and thepercentages of lung lobes with lesions were recorded on the NecropsyReport Form.

From each of the pigs necropsied at the second study site, a tissuesample of tonsil, lung, heart, and mesenteric lymph node was placed intoa single container with buffered 10% formalin; while another tissuesample from the same aforementioned organs was placed into a Whirl-Pak®(M-Tech Diagnostics Ltd., Thelwall, UK) and each Whirl-Pak® was placedon ice. Each container was properly labeled. Sample collections wererecorded on the Necropsy Report Form. Afterwards, formalin-fixed tissuesamples and a Diagnostic Request Form were submitted for IHC testing.IHC testing was conducted in accordance with standard laboratoryprocedures for receiving samples, sample and slide preparation, andstaining techniques. Fresh tissues in Whirl-Paks® were shipped with icepacks to the Study Monitor for storage (−70°±10° C.) and possible futureuse.

Formalin-fixed tissues were examined by a pathologist for detection ofPCV2 by IHC and scored using the following scoring system: 0=None;1=Scant positive staining, few sites; 2=Moderate positive staining,multiple sites; and 3=Abundant positive staining, diffuse throughout thetissue. For analytical purposes, a score of 0 was considered “negative,”and a score of greater than 0 was considered “positive.”

Results

Results for this example are given below. It is noted that Pigs No. 46and 98 died on days 14 and 25 respectively. These deaths werecategorized as bleeding deaths. Pig No. 11 (Group 1) was panting withrapid respiration on Day 15. Otherwise, all pigs were normal forbehavior, respiration and cough during this observation period and nosystemic adverse events were noted with any groups. No injection sitereactions were noted following vaccination on Day 0. Followingvaccination on Day 14, seven (7) out of fourteen (14) Group 1 pigs(50.0%) had swelling with a score of “2” on Day 15. Four (4) out offourteen (14) Group 1 (28.6%) still had a swelling of “2” on Day 16.None of the other groups experienced injection site reactions followingeither vaccination.

Average daily weight gain (ADWG) results are presented below in Table18. Pigs No. 46 and 98 that died from bleeding were excluded from groupresults. Group 4, which received one dose of 16 ug vORF2—Carbopol, hadthe highest ADWG (1.16±0.26 lbs/day), followed by Groups 1, 2, 3, 5, 6,and 10 which had ADWGs that ranged from 1.07±0.23 lbs/day to 1.11±0.26lbs/day. Group 9 had the lowest ADWG (0.88±0.29 lbs/day), followed byGroups 8 and 7, which had ADWGs of 0.93±0.33 lbs/day and 0.99±0.44lbs/day, respectively.

TABLE 18 Summary of Group Average Daily Weight Gains (ADWG) ADWG -lbs/day (Day 25 to Day 50) or adjusted for pigs Group Treatment N deadbefore Day 50 1 rORF2 - 16 μg - IMS 1314 2 doses 14 1.08 ± 0.30 lbs/day2 vORF2 - 16 μg - Carbopol 2 doses 15 1.11 ± 0.16 lbs/day 3 rORF2 - 16μg - Carbopol 2 doses 15 1.07 ± 0.21 lbs/day 4 vORF2 - 16 μg - Carbopol1 dose 15 1.16 ± 0.26 lbs/day 5 rORF2 - 4 μg - Carbopol 1 dose 15 1.07 ±0.26 lbs/day 6 rORF2 - 1 μg - Carbopol 2 doses 15 1.11 ± 0.26 lbs/day 7rORF2 - 0.25 μg - Carbopol 15 0.99 ± 0.44 lbs/day 2 doses 8 KV >8.0log - Carbopol 2 doses 15 0.93 ± 0.33 lbs/day 9 Challenge Controls 140.88 ± 0.29 lbs/day 10 Strict Negative Controls 15 1.07 ± 0.23 lbs/dayvORF2 = isolated viral ORF2; rORF2 = recombinant baculovirus expressedORF2; KV or killed whole cell virus = PCV2 virus grown in suitable cellculture

PVC2 serology results are presented below in Table 19. All ten (10)groups were seronegative for PCV2 on Day −3. On Day 14, PCV2 titersremained low for all ten (10) groups (range of 50-113). On Day 25, Group8, which received the whole cell killed virus vaccine, had the highestPCV2 titer (4617), followed by Group 2, which received 16 ugvORF2—Carbopol, Group 4, which received as single dose of 16 ugvORF2—Carbopol, and Group 3, which received 16 ug rORF2—Carbopol, whichhad titers of 2507, 1920 and 1503 respectively. On Day 32 (one week postchallenge), titers for Groups 1-6 and Group 8 ranged from 2360 to 7619;while Groups 7 (0.25 ug rORF2—Carbopol), 9 (Challenge Control), and 10(Strict negative control) had titers of 382, 129 and 78 respectively. OnDay 50 (day of necropsy), all ten (10) groups demonstrated high PCV2titers (>1257).

On Days 25, 32, and 50, Group 3, which received two doses of 16 ugrORF2—Carbopol had higher antibody titers than Group 1, which receivedtwo doses of 16 ug rORF2—IMS 1314. On Days 25, 32 and 50, Group 2, whichreceived two doses of 16 ug vORF2 had higher titers than Group 4, whichreceived only one does of the same vaccine. Groups 3, 5, 6, 7, whichreceived decreasing levels of rORF2—Carbopol, of 16, 4, 1, and 0.25 ugrespectively, demonstrated correspondingly decreasing antibody titers onDays 25 and 32.

TABLE 19 Summary of Group PCV2 IFA Titers Day Day Day Day Day GroupTreatment −3 14** 25*** 32 50**** 1 rORF2 - 16 μg - 50 64 646 3326 4314IMS 1314 2 doses 2 vORF2 - 16 μg - 50 110 2507 5627 4005 Carbopol 2doses 3 rORF2 - 16 μg - 50 80 1503 5120 6720 Carbopol 2 doses 4 vORF2 -16 μg - 50 113 1920 3720 1257 Carbopol 1 dose 5 rORF2 - 4 μg - 50 611867 3933 4533 Carbopol 1 dose 6 rORF2 - 1 μg - 50 70 490 2360 5740Carbopol 2 doses 7 rORF2 - 0.25 μg - 50 73 63 382 5819 Carbopol 2 doses8 KV > 8.0 log - 50 97 4617 7619 10817 Carbopol 2 doses 9 Challenge 5053 50 129 4288 Controls 10 Strict Negative 50 50 50 78 11205 ControlsvORF2 = isolated viral ORF2; rORF2 = recombinant baculovirus expressedORF2; KV or killed whole cell virus = PCV2 virus grown in suitable cellculture *For calculation purposes, a ≦100 IFA titer was designated as atiter of “50”; a ≧6400 IFA titer was designated as a titer of “12,800”.**Day of Challenge ***Day of Necropsy

The results from the post-challenge clinical observations are presentedbelow. Table 20 includes observations for Abnormal Behavior, AbnormalRespiration, Cough and Diarrhea. Table 21 includes the results from theSummary of Group Overall Incidence of Clinical Symptoms and Table 22includes results from the Summary of Group Mortality RatesPost-challenge. The incidence of abnormal behavior, respiration andcough post-challenge were low in pigs receiving 16 ug rORF2—IMS 1314(Group 1), 16 ug rORF2—Carbopol (Group 3), 1 ug rORF2—Carbopol (Group6), 0.25 ug rORF2—Carbopol (Group 7), and in pigs in the ChallengeControl Group (Group 9). The incidence of abnormal behavior respirationand cough post-challenge was zero in pigs receiving 16 ug vORF2—Carbopol(Group 2), a single dose of 16 ug vORF2—Carbopol (Group 4), 4 ugrORF2—Carbopol (Group 5), >8 log KV—Carbopol (Group 8), and in pigs inthe strict negative control group (Group 10).

The overall incidence of clinical symptoms varied between groups. Pigsreceiving 16 ug vORF2—Carbopol (Group 2), a single dose of 16 ugvORF2—Carbopol (Group 4), and pigs in the Strict negative control group(Group 10) had incidence rates of 0%; pigs receiving 16 ugrORF2—Carbopol (Group 3), and 1 ug rORF2—Carbopol (Group 6) hadincidence rates of 6.7%; pigs receiving 16 ug rORF2—IMS 1314 (Group 1)had an overall incidence rate of 7.1%; pigs receiving 4 ugrORF2—Carbopol (Group 5), 0.25 ug rORF2—Carbopol (Group 7), and >8 logKV vaccine had incidence rates of 13.3%; and pigs in the ChallengeControl Group (Group 9) had an incidence rate of 14.3%.

Overall mortality rates between groups varied as well. Group 8, whichreceived 2 doses of KV vaccine had the highest mortality rate of 20.0%;followed by Group 9, the challenge control group, and Group 7, whichreceived 0.25 ug rORF2—Carbopol and had mortality rates of 14.3% and13.3% respectively. Group 4, which received one dose of 16 ugvORF2—Carbopol had a 6.7% mortality rate. All of the other Groups, 1, 2,3, 5, 6, and 10 had a 0% mortality rate.

TABLE 20 Summary of Group Observations for Abnormal Behavior, AbnormalRespiration, and Cough Post-Challenge Abnormal Abnormal Group TreatmentN Behavior¹ Behavior² Cough³ 1 rORF2 - 16 μg - 14 0/14 0/14 1/14 IMS1314 2 doses (0%) (0%) (7.1%)   2 vORF2 - 16 μg - Carbopol 15 0/15 0/150/15 2 doses (0%) (0%) (0%) 3 rORF2 - 16 μg - Carbopol 15 0/15 0/15 1/152 doses (0%) (0%) (6.7%)   4 vORF2 - 16 μg - Carbopol 15 0/15 0/15 0/151 dose (0%) (0%) (0%) 5 rORF2 - 4 μg - 15 1/15 1/15 0/15 Carbopol 1 dose(6.7%)   (6.7%)   (0%) 6 rORF2 - 1 μg - 15 0/15 0/15 1/15 Carbopol 2doses (0%) (0%) (6.7%)   7 rORF2 - 0.25 μg - 15 0/15 1/15 1/15 Carbopol2 doses (0%) (6.7%)   (06.7%)   8 KV >8.0 log - Carbopol 2 15 1/15 1/150/15 doses (6.7%)   (6.7%)   (0%) 9 Challenge Controls 14 1/14 1/14 2/14(7.1%)   (7.1%)   (14/3%)   10 Strict Negative Controls 15 0/15 0/150/15 (0%) (0%) (0%) ¹Total number of pigs in each group thatdemonstrated any abnormal behavior for at least one day ²Total number ofpigs in each group that demonstrated any abnormal respiration for atleast one day ³Total number of pigs in each group that demonstrated acough for at least one day

TABLE 21 Summary of Group Overall Incidence of Clinical Symptoms Post -Challenge Incidence of pigs with Clinical Incidence Group Treatment NSymptoms¹ Rate 1 rORF2 - 16 μg - 14 1 7.1% IMS 1314 2 doses 2 vORF2 - 16μg - Carbopol 2 15 0 0.0% doses 3 rORF2 - 16 μg - Carbopol 2 15 1 6.7%doses 4 vORF2 - 16 μg - Carbopol 1 15 0 0.0% dose 5 rORF2 - 4 μg - 15 213.3% Carbopol 1 dose 6 rORF2 - 1 μg - 15 1 6.7% Carbopol 2 doses 7rORF2 - 0.25 μg - Carbopol 15 2 13.3% 2 doses 8 KV >8.0 log - Carbopol 215 2 13.3% doses 9 Challenge Controls 14 2 14.3% 10 Strict NegativeControls 15 0 0.0% vORF2 = isolated viral ORF2; rORF2 = recombinantbaculovirus expressed ORF2; KV or killed whole cell virus = PCV2 virusgrown in suitable cell culture ¹Total number of pigs in each group thatdemonstrated any clinical symptom for at least one day

TABLE 22 Summary of Group Mortality Rates Post-Challenge Dead Post-Mortality Group Treatment N challenge Rate 1 rORF2 - 16 μg - 14 0 0.0%IMS 1314 2 doses 2 vORF2 - 16 μg - Carbopol 2 15 0 0.0% doses 3 rORF2 -16 μg - Carbopol 2 15 0 0.0% doses 4 vORF2 - 16 μg - Carbopol 1 15 16.7% dose 5 rORF2 - 4 μg - 15 0 0.0% Carbopol 1 dose 6 rORF2 - 1 μg - 150 0.0% Carbopol 2 doses 7 rORF2 - 0.25 μg - Carbopol 2 15 2 13.3% doses8 KV >8.0 log - Carbopol 2 15 3 20.0% doses 9 Challenge Controls 14 214.3% 10 Strict Negative Controls 15 0 0.0% vORF2 = isolated viral ORF2;rORF2 = recombinant baculovirus expressed ORF2; KV or killed whole cellvirus = PCV2 virus grown in suitable cell culture

The Summary of Group Mean Percentage Lung Lesions and TentativeDiagnosis is given below in Table 23. Group 9, the challenge controlgroup, had the highest percentage lung lesions with a mean of10.81±23.27%, followed by Group 7, which received 0.25 ug rORF2—Carbopoland had a mean of 6.57±24.74%, Group 5, which received 4 ugrORF2—Carbopol and had a mean of 2.88±8.88%, and Group 8, which receivedthe KV vaccine and had a mean of 2.01±4.98%. The remaining six (6)groups had lower mean percentage lung lesions that ranged from0.11±0.38% to 0.90±0.15%.

Tentative diagnosis of pneumonia varied among the groups. Group 3, whichreceived two doses of 16 ug rORF2—Carbopol, had the lowest tentativediagnosis of pneumonia, with 13.3%. Group 9, the challenge controlgroup, had 50% of the group tentatively diagnosed with pneumonia,followed by Group 10, the strict negative control group and Group 2,which received two doses of 16 ug vORF2—Carbopol, with 46.7% of 40%respectively, tentatively diagnosed with pneumonia.

Groups 1, 2, 3, 5, 9, and 10 had 0% of the group tentatively diagnosedas PCV2 infected; while Group 8, which received two doses if KV vaccine,had the highest group rate of tentative diagnosis of PCV2 infection,which 20%. Group 7, which received two doses of 0.25 ug rORF2—Carbopol,and Group 4, which received one dose of 16 ug vORF2—Carbopol hadtentative group diagnoses of PCV2 infection in 13.3% and 6.7% of eachgroup, respectively.

Gastric ulcers were only diagnosed in one pig in Group 7 (6.7%); whilethe other 9 groups remained free of gastric ulcers.

TABLE 23 Summary of Group Mean % Lung Lesion and Tentative Diagnosis No.Of pigs that shed for Incidence Group Treatment N at least one day Rate1 rORF2 - 16 μg - 15 0   0% IMS 1314 2 doses 2 vORF2 - 16 μg - 15 1 6.7% Carbopol 2 doses 3 rORF2 - 16 μg - 15 3 20.0% Carbopol 2 doses 4vORF2 - 16 μg - 15 2 13.3% Carbopol 1 dose 5 rORF2 - 4 μg - 15 3 20.0%Carbopol 1 dose 6 rORF2 - 1 μg - 15 6 40.0% Carbopol 2 doses 7 rORF2 -0.25 μg - 15 7 46.7% Carbopol 2 doses 8 KV >8.0 log - Carbopol 15 12  80% 2 doses 9 Challenge Controls 14 14 100.0%  10 Strict NegativeControls 15 14 93.3% vORF2 = isolated viral ORF2; rORF2 = recombinantbaculovirus expressed ORF2; KV or killed whole cell virus = PCV2 virusgrown in suitable cell culture

The Summary of Group IHC Positive Incidence Results are shown below inTable 24. Group 1 (16 ug rORF2—IMS 1314) had the lowest group rate ofIHC positive results with 0% of the pigs positive for PCV2, followed byGroup 2 (16 ug vORF2—Carbopol) and Group 4 (single dose 16 ugvORF2—Carbopol), which had group IHC rates of 6.7% and 13.3%respectively. Group 9, the challenge control group, had the highest IHCpositive incidence rate with 100% of the pigs positive for PCV2,followed by Group 10, the strict negative control group, and Group 8 (KVvaccine), with 93.3% and 80% of the pigs positive for PCV2,respectively.

TABLE 24 Summary of Group IHC Positive Incidence Rate No. Of pigs thatshed for Group Treatment N at least one day Incidence Rate 1 rORF2 - 16μg - 15 0   0% IMS 1314 2 doses 2 vORF2 - 16 μg - 15 1  6.7% Carbopol 2doses 3 rORF2 - 16 μg - 15 3 20.0% Carbopol 2 doses 4 vORF2 - 16 μg - 152 13.3% Carbopol 1 dose 5 rORF2 - 4 μg - 15 3 20.0% Carbopol 1 dose 6rORF2 - 1 μg - 15 6 40.0% Carbopol 2 doses 7 rORF2 - 0.25 μg - 15 746.7% Carbopol 2 doses 8 KV >8.0 log - Carbopol 15 12   80% 2 doses 9Challenge Controls 14 14 100.0%  10 Strict Negative Controls 15 14 93.3%vORF2 = isolated viral ORF2; rORF2 = recombinant baculovirus expressedORF2; KV or killed whole cell virus = PCV2 virus grown in suitable cellculture

Discussion

Seven PCV2 vaccines were evaluated in this example, which included ahigh dose (16 μg) of rORF2 antigen adjuvanted with IMS 1314 administeredtwice, a high dose (16 μg) of vORF2 antigen adjuvanted with Carbopoladministered once to one group of pigs and twice to a second group ofpigs, a high dose (16 μg) of rORF2 antigen adjuvanted with Carbopoladministered twice, a 4 μg dose of rORF2 antigen adjuvanted withCarbopol administered twice, a 1 μg dose of rORF2 antigen adjuvantedwith Carbopol administered twice, a low dose (0.25 μg) of rORF2 antigenadjuvanted with Carbopol administered twice, and a high dose (>8 log) ofkilled whole cell PCV2 vaccine adjuvanted with Carbopol. Overall, Group1, which received two doses of 16 μg rORF2—IMS 1314, performed slightlybetter than Groups 2 through 7, which received vaccines containingvarious levels of either vORF2 or rORF2 antigen adjuvanted with Carbopoland much better than Group 8, which received two doses of killed wholecell PCV2 vaccine. Group 1 had the third highest ADWG (1.80±0.30lbs/day), the lowest incidence of abnormal behavior (0%), the lowestincidence of abnormal respiration (0%), a low incidence of cough (7.1%),a low incidence of overall clinical symptoms (7.1%), was tied with threeother groups for the lowest mortality rate (0%), the second lowest ratefor mean % lung lesions (0.15±0.34%), the second lowest rate forpneumonia (21.4%) and the lowest incidence rate for positive IHC tissues(0%). Group 1 was, however, the only group in which injection sitereactions were noted, which included 50% of the vaccinates 1 day afterthe second vaccination. The other vaccines administered to Groups 2through 7 performed better than the killed vaccine and nearly as well asthe vaccine administered to Group 1.

Group 8, which received two doses of killed PCV2 vaccine adjuvanted withCarbopol, had the worst set of results for any vaccine group. Group 8had the lowest ADWG (0.93±0.33 lbs/day), the second highest rate ofabnormal behavior (6.7%), the highest rate of abnormal respiration(6.7%), was tied with three other groups for the highest overallincidence rate of clinical symptoms (13.3%), had the highest mortalityrate of all groups (20%), and had the highest positive IHC rate (80%) ofany vaccine group. There was concern that the killed whole cell PCV2vaccine may not have been fully inactivated prior to administration toGroup 8, which may explain this group's poor results. Unfortunately,definitive data was not available to confirm this concern. Overall, inthe context of this example, a Conventional Killed PCV2 vaccine did notaid in the reduction of PCV2 associated disease.

As previously mentioned, no adverse events were associated with the testvaccines with exception of the vaccine adjuvanted with IMS 1314.Injection site reactions were noted in 50.0% of the pigs 1 day after thesecond vaccination with the vaccine formulated with IMS 1314 and in28.6% of the pigs 2 days after the second vaccination. No reactions werenoted in any pigs receiving Carbopol adjuvanted vaccines. Any furtherstudies that include pigs vaccinated with IMS 1314 adjuvanted vaccinesshould continue to closely monitor pigs for injection site reactions.

All pigs were sero-negative for PCV2 on Day −3 and only Group 2 had atiter above 100 on Day 14. On Day 25 (day of challenge), Group 8 had thehighest PCV2 antibody titer (4619), followed by Group 2 (2507). With theexception of Groups 7, 9 and 10, all groups demonstrated a strongantibody response by Day 32. By Day 50, all groups including Groups 7, 9and 10 demonstrated a strong antibody response.

One of the hallmarks of late stage PCV2 infection and subsequent PMWSdevelopment is growth retardation in weaned pigs, and in severe cases,weight loss is noted. Average daily weight gain of groups is aquantitative method of demonstrating growth retardation or weight loss.In this example, there was not a large difference in ADWG betweengroups. Group 8 had the lowest ADWG of 0.88±0.29 lbs/day, while Group 4had the highest ADWG of 1.16±0.26 lb/day. Within the context of thisstudy there was not a sufficient difference between groups to basefuture vaccine efficacy on ADWG.

In addition to weight loss—dyspnea, lethargy, pallor of the skin andsometimes icterus are clinical symptoms associated with PMWS. In thisexample, abnormal behavior and abnormal respiration and cough were notedinfrequently for each group. As evidenced in this study, this challengemodel and challenge strain do not result in overwhelming clinicalsymptoms and this is not a strong parameter on which to base vaccineefficacy.

Overall, mortality rates were not high in this example and the lack of ahigh mortality rate in the challenge control group limits this parameteron which to base vaccine efficacy. Prior to Day 46, Groups 4 and 7 eachhad one out of fifteen pigs die, Group 9 had two out of fourteen pigsdie and Group 8 had three out of fifteen pigs die. Due to the fact thatGroup 9, the challenge control group was not demonstrating PCV2 clinicalsymptoms and only two deaths had occurred in this group by Day 46,Porcine Respiratory and Reproductive Syndrome Virus (PRRSV) MLV vaccinewas administered to all pigs on Day 46. Earlier studies had utilizedINGELVAC® PRRS MLV as an immunostimulant to exasperate PCV2-associatedPMWS disease and mortality rates were higher in these earlier studies.Two deaths occurred shortly after administering the PRRS vaccine on Day46—Group 4 had one death on Day 46 and Group 7 had one death on Day47—which were probably not associated with the administration of thePRRS vaccine. By Day 50, Group 8, which received two doses of killedvaccine, had the highest mortality rate (20%), followed by Group 9(challenge control) and Group 7 (0.25 ug rORF2—Carbopol), with mortalityrates of 14.3% and 13.3% respectively. Overall, administration of thePRRS vaccine to the challenge model late in the post-challengeobservation phase of this example did not significantly increasemortality rates.

Gross lesions in pigs with PMWS secondary to PCV2 infection typicallyconsist of generalized lymphadenopathy in combination with one or moreof the following: (1) interstitial pneumonia with interlobular edema,(2) cutaneous pallor or icterus, (3) mottled atrophic livers, (4)gastric ulcers and (5) nephritis. At necropsy (Day 50), icterus,hepatitis, and nephritis were not noted in any groups. A gastric ulcerwas noted in one Group 7 pig, but lymphadenopathy was not specificallyexamined for. Based on the presence of lesions that were consistent withPCV2 infection, three groups had at least one pig tentatively diagnosedwith PCV2 (PMWS). Group 8, which received two doses of killed vaccine,had 20% tentatively diagnosed with PCV2, while Group 7 and Group 4 had13.3% and 6.7%, respectively, tentatively diagnosed with PCV2. The mean% lung lesion scores varied between groups at necropsy. Groups 1, 2, 3,4, 6 and 10 had low % lung lesion scores that ranged from 0.11±0.38% to0.90±0.15%. As expected, Group 9, the challenge control group, had thehighest mean % lung lesion score (10.81±23.27%). In four groups, themean % lung lesion scores were elevated due to one to three pigs in eachof these groups having very high lung lesion scores. The lung lesionswere red/purple and consolidated. Typically, lung lesions associatedwith PMWS are described as tan, non-collapsible with interlobular edema.The lung lesions noted in this study were either not associated withPCV2 infection or a second pulmonary infectious agent may have beenpresent. Within the context of this study, the % lung lesion scoresprobably do no reflect a true measure of the amount of lung infectiondue to PCV2. Likewise, tentative diagnosis of pneumonia may have beenover-utilized as well. Any pigs with lung lesions, some as small as0.10% were listed with a tentative diagnosis of pneumonia. In thisexample, there was no sufficient difference between groups with respectto gross lesions and % lung lesions on which to base vaccine efficacy.

IHC results showed the largest differences between groups. Group 1 (16μg rORF2—IMS 1314) had the lowest positive IHC results for PCV2 antigen(0%); while Groups 9 and 10 had the highest positive IHC results withincidence rates of 100% and 93.3% respectively. Groups 3, 5, 6 and 7,which received 16, 4, 1 or 0.25 μg of rORF2 antigen, respectively,adjuvanted with Carbopol, had IHC positive rates of 20%, 20%, 40% and46.7%, respectively. Group 2, which received two doses of 16 μg vORF2adjuvanted with Carbopol had an IHC positive rate of 6.7%, while Group 4which received only one dose of the same vaccine, had an IHC positiverate of 13.3%. Due to the objective nature of this test and the factthat IHC results correlated with expected results, IHC testing isprobably one of the best parameters on which to base vaccine efficacy.

Thus in one aspect of the present invention, the Minimum ProtectiveDosage (MPD) of PCV2 rORF2 antigen adjuvanted with Carbopol in the CDCDpig model in the face of a PCV2 challenge is determined Groups 3, 5, 6and 7 each received two doses of rORF2 antigen adjuvanted with Carbopol,but the level of rORF2 antigen varied for each group. Groups 3, 5, 6 and7 each received 16, 4, 1 or 0.25 μg of rORF2 antigen respectively. Ingeneral, decreasing the level of rORF2 antigen decreased PCV2 antibodytiters, and increased the mortality rate, mean % lung lesions and theincidence of IHC positive tissues. Of the four groups receiving varyinglevels of rORF2—Carbopol, Groups 3 and 5, which received two doses of 16or 4 μg of rORF2 antigen, respectively, each had an IHC positive rate ofonly 20%, and each had similar antibody titers. Overall, based on IHCpositive results, the minimum protective dosage of rORF2 antigenadministered twice is approximately 4 μg.

In another aspect of the present invention, the antigenicity ofrecombinant (rORF2) and VIDO R-1 (vORF2) PCV2 antigens were assessed.Group 2 received two doses of 16 μg vORF2 and Group 3 received two dosesof 16 μg rORF2. Both vaccines were adjuvanted with Carbopol. Bothvaccines were found to be safe and both had 0% mortality rate. Group 2had a PCV2 antibody titer of 2507 on Day 25, while Group 3 had a PCV2antibody titer of 1503. Group 3 had a lower mean % lung lesion scorethan Group 2 (0.11±0.38% vs. 0.90±0.15%), but Group 2 had a lower IHCpositive incidence rate that Group 3 (6.7% vs. 20%). Overall, bothvaccines had similar antigenicity, but vORF2 was associated withslightly better IHC results.

In yet another aspect of the present invention, the suitability of twodifferent adjuvants (Carbopol and IMS 1314) was determined Groups 1 and3 both received two doses of vaccine containing 16 ug of rORF2 antigen,but Group 1 received the antigen adjuvanted with IMS 1314 while Group 3received the antigen adjuvanted with Carbopol. Both groups hadessentially the same ADWG, essentially the same incidence of clinicalsigns post-challenge, the same mortality rate, and essentially the samemean % lung lesions; but Group 1 had an IHC positive rate of 0% whileGroup 3 had an IHC positive rate of 20%. However, Group 3, whichreceived the vaccine adjuvanted with Carbopol had higher IFAT PCV2titers on Days 25, 32 and 50 than Group 1, which received the vaccineadjuvanted with IMS 1314. Overall, although the PCV2 vaccine adjuvantedwith IMS 1314 did provide better IHC results, it did not provideoverwhelmingly better protection from PCV2 infection and did induceinjection site reaction. Whereas the PCV2 vaccine adjuvanted withCarbopol performed nearly as well as the IMS 1314 adjuvanted vaccine,but was not associated with any adverse events.

In still another aspect of the present invention, the feasibility ofPCV2 ORF2 as a 1 ml, 1 dose product was determined Groups 2 and 4 bothreceived 16 μg of vORF2 vaccine adjuvanted with Carbopol on Day 0, butGroup 2 received a second dose on Day 14. Group 4 had a slightly higherADWG and a lower mean % lung lesions than Group 2, but Group 2 hadhigher IFAT PCV2 titers on Day 25, 32 and 50, and a slightly lowerincidence rate of IHC positive tissues. All other results for these twogroups were similar. Overall, one dose of vORF2 adjuvanted with Carbopolperformed similar to two doses of the same vaccine.

What is claimed is:
 1. A method of recovering recombinant proteinexpressed by open reading frame 2 of PCV2 comprising the steps of: A)cloning said recombinant open reading frame 2 from PCV2 into a transfervector; B) transfecting the portion of said transfer vector containingsaid recombinant open reading frame 2 into a virus; C) infecting cellsin media with said virus; D) causing said virus to express the proteinfrom said open reading frame 2; E) separating cells from said vectorfrom the supernate; and F) recovering said expressed open reading frame2 protein in said supernate.
 2. The method of claim 1, said methodfurther including the step of amplifying said open reading frame 2 froma strain of PCV2 prior to cloning said open reading frame into saidtransfer vector.
 3. The method of claim 1, said recombinant open readingframe 2 further comprising a sequence selected from the group consistingof a 5′ Kozak's sequence, a 3′ EcoR1 site, and combinations thereof. 4.The method of claim 3, said 5′ Kozak's sequence comprising SEQ ID NO: 1.5. The method of claim 3, wherein said 3′ EcoR1 site comprises SEQ IDNO:
 2. 6. The method of claim 1, wherein said PCV2 open reading framecomprises SEQ ID NO:
 4. 7. The method of claim 1, wherein saidrecombinant protein comprises SEQ ID No:
 6. 8. The method of claim 1,wherein said media comprises serum-free insect cell media.
 9. The methodof claim 1, further comprising the steps of: i) prior to step A, cloningsaid amplified open reading frame 2 into a first vector; ii) excisingsaid open reading frame 2 from said first vector; and iii) using saidexcised open reading frame 2 in step A.
 10. The method of claim 1,wherein said cells comprise SF+ cells.
 11. The method of claim 1,wherein said virus comprises baculovirus.
 12. The method of claim 1,wherein said transfected portion comprises SEQ ID NO:
 4. 13. The methodof claim 1, said open reading frame 2 protein being recovered in saidsupernate at least 5 days after infecting the cells with said virus. 14.A method of preparing a composition for invoking an immune responseagainst PCV2, said method comprising the steps of: i) transfecting aconstruct into a virus, said construct comprising recombinant DNA fromopen reading frame 2 of PCV2; ii) infecting cells with said transfectedvirus, said cells being in a growth media; iii) causing said virus toexpress the recombinant protein from said open reading frame 2; iv)recovering said expressed open reading frame 2 protein in the supernate;and v) combining said recovered protein with a suitable adjuvant orother pharmaceutically acceptable carrier or excipient.
 15. The methodof claim 14, wherein said method further including the step of obtainingsaid construct from a transfer vector.
 16. The method of claim 14, saidmethod further including the step of amplifying said open reading frame2 from a strain of PCV2 prior to cloning said open reading frame 2 intosaid transfer vector.
 17. The method of claim 14, said recombinant openreading frame 2 further comprising a sequence selected from the groupconsisting of a 5′ Kozak's sequence, a 3′ EcoR1 site, and combinationsthereof.
 18. The method of claim 17, said 5′ Kozak's sequence comprisingSEQ ID No:
 1. 19. The method of claim 17, said 3′ EcoR1 site comprisingSEQ ID No:
 2. 20. The method of claim 14, said PCV2 open reading frame 2comprising SEQ ID NO:
 4. 21. The method of claim 14, said recombinantprotein comprising SEQ ID NO:
 6. 22. The method of claim 14, said mediacomprising serum-free insect cell media.
 23. The method of claim 16,further comprising the steps of: i) cloning said amplified open readingframe 2 into a first vector; ii) excising said open reading frame 2 fromsaid first vector; and using said excised open reading frame 2 forcloning into said transfer vector.
 24. The method of claim 14, saidcells comprising SF+ cells.
 25. The method of claim 14, said viruscomprising baculovirus.
 26. The method of claim 14, said transfectedconstruct comprising SEQ ID NO:
 4. 27. The method of claim 14, said openreading frame 2 protein being recovered at least 5 days after infectingsaid cells with said virus.
 28. The method of claim 14, said recoverystep further comprising the step of separating said media from saidcells and cell debris.
 29. The method of claim 28, said separating stepincluding the step of filtering said cells, cell debris, and growthmedia through a filter having pores ranging in size from about 0.45 μMto about 1.0 μM.
 30. The method of claim 14, said method furtherincluding the step of inactivating said virus prior to combining saidrecovered protein with a suitable adjuvant.
 31. A method of recoveringthe protein expressed by open reading frame 2 from PCV2, said methodincluding the steps i) infecting cells in growth media with arecombinant viral vector containing said open reading frame 2; ii)causing said vector to express said protein; and iii) recovering saidexpressed protein in the supernate.
 32. The method of claim 31, saidrecovering occurring at least 5 days after infection of the cells withsaid viral vector.
 33. A composition of matter obtained by a methodselected from the group consisting of the methods of claim 1, claim 14,claim 31, and combinations thereof.
 34. The use of the composition ofclaim 33 as a medicament.
 35. The use of the composition of claim 33 forthe preparation of a medicament for the prevention of a PCV2 infection.36. A method for producing a diagnostic kit for the detection of a PCV2infection in a sample which comprises i) producing a recombinant proteinusing a method selected from the group consisting of the methods ofclaim 1, claim 14, claim 31, and combinations thereof; ii) and packagingsaid recombinant protein into a suitable container.
 37. The method ofclaim 36, further comprising the step of including an instruction manualwith the container that packages the recombinant protein.
 38. Animmunogenic composition effective for lessening the severity of clinicalsymptoms associated with PCV2 infection comprising PCV2 ORF2 protein, oran immunogenic portion thereof, wherein said immunogenic portion has atleast 10 contiguous amino acids from PCV2 ORF2 protein.
 39. Theimmunogenic composition of claim 38, wherein said PCV2 ORF2 protein isi) a polypeptide comprising a sequence selected form the groupconsisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 orSEQ ID NO: 11; ii) any polypeptide that is at least 80% homologous tothe polypeptide of i), iii) any immunogenic portion of the polypeptidesof i) and/or ii) iv) the immunogenic portion of comprising at least 10contiguous amino acids included in the sequences of SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID No: 9, SEQ ID No: 10 or SEQ ID No: 11, v) a polypeptidethat is encoded by a DNA comprising the sequence of SEQ ID NO: 3 or SEQID NO:
 4. vi) any polypeptide that is encoded by a polynucleotide thatis at least 80% homologous to the polynucleotide of v), vii) anyimmunogenic portion of the polypeptides encoded by the polynucleotide ofv) and/or vi) viii) the immunogenic portion of vii), wherein thepolynucleotide coding for said immunogenic portion comprises at least 30contiguous nucleotides included in the sequences of SEQ ID No: 3, or SEQID No:
 4. 40. The immunogenic composition of claim 38, furthercomprising an inactivated viral vector and cell culture supernate. 41.The immunogenic composition of claim 40, wherein said inactivated viralvector is a recombinant baculovirus coding for the PCV2 ORF2 protein.42. The immunogenic composition of claim 40, wherein the compositioncomprises BEI.
 43. The immunogenic composition of claim 42, wherein saidcomposition comprises sodium thiosulfate.
 44. The immunogeniccomposition of claim 39, comprising an additional ingredient selectedfrom the group consisting of carriers, adjuvants, media, viralinactivators, diluents, isotonic agents, immunomodulatory agents,antibiotics, and combinations thereof.
 45. The immunogenic compositionof claim 44, wherein said composition comprises an adjuvant, preferablycarbopol.
 46. The immunogenic composition of claim 45, wherein saidcomposition comprises a pharmaceutical acceptable salt, preferablysaline.
 47. The immunogenic composition of claim 38, wherein saidcomposition comprises at least 0.2 mcg/ml PCV2 ORF2 protein.
 48. Theimmunogenic composition of claim 38, wherein said composition comprisesat least 2 μg PCV2 ORF2 protein.
 49. A container comprising at least onedose of the immunogenic composition of claim 38, wherein one dosecomprises at least 2 μg PCV2 ORF2 protein.
 50. A container comprising 10to 250 doses of the immunogenic composition of claim 38, wherein onedose comprises at least 2 μg PCV2 ORF2 protein.
 51. The container ofclaim 49, further comprising an anti-microbiological active agent.
 52. Akit comprising a container of claim 49 and an instruction manual,including the information for the intramuscular application of at leastone dose of the immunogenic composition into piglets for lessening theseverity of clinical symptoms associated with PCV2 infection.
 53. Thekit of claim 52, wherein the instruction manual comprises theinformation of a second or further administration(s) of at least onedose of the immunogenic composition, wherein the second administrationor any further administration is at least 14 days beyond the lastadministration.